Compositions that treat or inhibit pathological conditions associated with inflammatory response

ABSTRACT

A natural formulation of compounds that would to modulate inflammation is disclosed. The formulation would also inhibit expression of COX-2, inhibit synthesis of prostaglandins selectively in target cells, and inhibit inflammatory response selectively in target cells. The compositions containing at least one fraction isolated or derived from hops. Other embodiments relate to combinations of components, including at least one fraction isolated or derived from hops, tryptanthrin and conjugates thereof, rosemary, an extract or compound derived from rosemary, a triterpene species, or a diterpene lactone or derivatives or conjugates thereof.

RELATED APPLICATIONS

This application is the U.S. National Phase under 35 U.S.C. §371 ofInternational Application No. PCT/US03/33362, filed on Oct. 20, 2003,which claims priority to U.S. application Ser. No. 10/464,410, filed onJun. 18, 2003 (now U.S. Pat. No. 8,142,819), and U.S. application Ser.No. 10/464,834, filed on Jun. 18, 2003 (now abandoned), and U.S.application Ser. No. 10/400,293, filed on Mar. 26, 2003 (now abandoned),and U.S. application Ser. No. 10/401,283, filed on Mar. 26, 2003 (nowabandoned), and U.S. Provisional Application No. 60/450,237, filed Feb.25, 2003 (now expired), and U.S. Provisional Application No. 60/420,383,filed Oct. 21, 2002 (now expired). The disclosures of each of the aboveApplications is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to compositions that can be usedto treat or inhibit pathological conditions associated withtissue-specific activation of inflammation and/or NFκB, to methods ofmodulating inflammation, including in cells, and to methods ofmodulating NFκB in cells. More specifically, the invention relates to acomposition comprising hops extracts or derivatives thereof or afraction isolated or derived from hops, which can optionally be combinedwith a second component, such as rosemary, an extract derived fromrosemary, a compound derived from rosemary, a triterpene species, aditerpene lactone species, and tryptanthrin. The invention furtherrelates to methods of using the compositions to inhibit expression ofcyclooxygenase-2 (COX-2), inhibit synthesis of prostaglandinsselectively in target cells, inhibit inflammatory responses selectivelyin target cells, and/or inhibit NFκB activation selectively in targetcells.

2. Description of the Related Art

Cyclooxygenase (prostaglandin endoperoxide synthase, EC 1.14.991, COX)catalyzes the rate-limiting step in the metabolism of arachidonic acidto prostaglandin H₂ (PGH₂), which is further metabolized to variousprostaglandins, prostacyclin and thromboxane A2 (c.f. FIG. 1). In theearly 1990s, it was established that COX exists in two isoforms,commonly referred to as COX-1 and COX-2. It was subsequently determinedthat the COX-1 and COX-2 proteins are derived from distinct genes thatdiverged well before birds and mammals. Prostaglandins (PGs) generatedvia the COX-1 and COX-2 pathways are identical molecules and thereforehave identical biological effects. COX-1 and COX-2, however, maygenerate a unique pattern and variable amounts of eicosanoids;therefore, relative differences in the activation of these isozymes mayresult in quite dissimilar biological responses. Differences in thetissue distribution and regulation of COX-1 and COX-2 are now consideredcrucial for the beneficial as well as adverse effects of COX inhibitors.

The generally held concept (COX dogma) is that COX-1 is expressedconstitutively in most tissues whereas COX-2 is the inducible enzymetriggered by pro-inflammatory stimuli including mitogens, cytokines andbacterial lipopolysaccharide (LPS) in cells in vitro and in inflamedsites in vivo. Based primarily on such differences in expression, COX-1has been characterized as a housekeeping enzyme and is thought to beinvolved in maintaining physiological functions such as cytoprotectionof the gastric mucosa, regulation of renal blood flow, and control ofplatelet aggregation. COX-2 is considered to mainly mediateinflammation, although constitutive expression is found in brain, kidneyand the gastrointestinal tract. Therefore, it would be desirable todown-regulate tissue-specific or cell-specific expression of COX-2.

Arachidonic acid serves as the primary substrate for the biosynthesis ofall PGs. PGs are ubiquitous hormones that function as both paracrine andautocrine mediators to affect a myriad of physiological changes in theimmediate cellular environment. The varied physiological effects of PGsinclude inflammatory reactions such as rheumatoid arthritis andosteoarthritis, blood pressure control, platelet aggregation, inductionof labor and aggravation of pain and fever. The discovery 30 years agothat aspirin and other non-steroidal analgesics inhibited PG productionidentified PG synthesis as a target for drug development. There are atleast 16 different PGs in nine different chemical classes, designatedPGA to PGI. PGs are part of a larger family of 20-carbon-containingcompounds called eicosanoids; they include prostacyclins, thromboxanes,and leukotrienes. The array of PGs produced varies depending on thedownstream enzymatic machinery present in a particular cell type. Forexample, endothelial cells produce primarily PGI₂, whereas plateletsmainly produce TXA₂.

Prostaglandins (PG) are believed to play an important role inmaintenance of human gastric mucosal homeostasis. Current dogma is thatCOX-1 is responsible for PG synthesis in normal gastric mucosa in orderto maintain mucosal homeostasis and that COX-2 is expressed by normalgastric mucosa at low levels, with induction of expression during ulcerhealing, following endotoxin exposure or cytokine stimulation. It nowappears that both COX-1 and COX-2 have important physiological roles inthe normal gastric mucosa.

Compounds that inhibit the production of PGs by COX have becomeimportant drugs in the control of pain and inflammation. Collectivelythese agents are known as non-steroidal anti-inflammatory drugs (NSAIDs)with their main indications being osteoarthritis and rheumatoidarthritis. However, the use of NSAIDs, and in particular aspirin, hasbeen extended to prophylaxis of cardiovascular disease. Over the lastdecade, considerable effort has been devoted to developing new moleculesthat are direct inhibitors of the enzymatic activity of COX-2, with theinference that these compounds would be less irritating to the stomachwith chronic use. Therefore, it would be desirable to inhibitinflammation response selectively in target cells.

U.S. patent application 2002/0086070A1 of Kuhrts entitled,“ANTI-INFLAMMATORY AND CONNECTIVE TISSUE REPAIR FORMULATIONS” describesa hops component that has an IC₅₀-WHMA COX-2/COX-1 ratio ranging fromabout 0.23 to about 3.33. Example 1 of the application describes acomposition containing an extract obtained through supercritical carbondioxide extraction of whole hops (CO₂-extract) comprising 42% humulone.

U.S. Pat. No. 6,391,346 entitled, “ANTI-INFLAMMATORY, SLEEP-PROMOTINGHERBAL COMPOSITION AND METHOD OF USE” describes an orally administeredcomposition capable of reducing inflammation in animals, while promotingsleep for such animals. The composition contains hydroalcoholic extractof hops and supercritical carbon dioxide extract of hops which are usedto promote sleep.

An ideal formulation for the treatment of inflammation would inhibit theinduction and activity of COX-2 without inhibiting the synthesis of PGE₂in gastric mucosal cells. However, conventional non-steroidalanti-inflammatory drugs lack the specificity of inhibiting COX-2 withoutaffecting gastric PGE₂ synthesis and are at risk to cause damages on thegastrointestinal system, when used for extended periods. Indeed, eventhe newly developed, anti-inflammatory drugs such as rofecoxib andcelexocib produce untoward gastric toxicity in the form of inducedspontaneous bleeding and delay of gastric ulcer healing.

Thus, it would be useful to identify a formulation of compounds thatwould specifically inhibit or prevent the synthesis of prostaglandins byCOX-2 with little or no effect on synthesis of PGE₂ in the gastricmucosa. Such a formulation, which would be useful for preserving thehealth of joint tissues, for treating arthritis or other inflammatoryconditions, has not previously been discovered. The term “specific orselective COX-2 inhibitor” was coined to embrace compounds or mixturesof compounds that selectively inhibit COX-2 over COX-1. However, whilethe implication is that such a calculated selectivity will result inlower gastric irritancy, unless the test materials are evaluated ingastric cells, the term “selective COX-2 inhibitor” does not carryassurance of safety to gastrointestinal cells. Only testing of compoundaction in target tissues, inflammatory cells and gastric mucosal cells,will identify those agents with low potential for stomach irritation.

The major problem associated with ascertaining COX-2 selectivity (i.e.low gastric irritancy) is that differences in assay methodology can haveprofound effects on the results obtained. Depicted in Table 1 are thecategories of the numerous in vitro assays that have been developed fortesting and comparing the relative inhibitory activities of NSAID andnatural compounds against COX-1 and COX-2. These test systems can beclassified into three groups: (1) systems using animal enzymes, animalcells or cell lines, (2) assays using human cell lines, or humanplatelets and monocytes, and (3) currently evolving models using humancells that are representative of the target cells for theanti-inflammatory and adverse effects of NSAID and dietary supplements.Generally, models using human cell lines or human platelets andmonocytes are the current standard and validated target cell models havenot been forthcoming. A human gastric cell line capable of assessingpotential for gastric irritancy is a need.

TABLE 1 Classification of test systems for in vitro assays assessingCOX-2 selectivity of anti-inflammatory compounds† TEST SYSTEMS ANIMALHUMAN TARGET Enzymes Enzymes Human Gastric Mucosa Cells Cells CellsHuman Chondrocytes Cell lines Cell lines Human Synoviocytes OTHER SYSTEMVARIABLES 1. Source of arachidonic acid - endogenous or exogenous; 2.Various expression systems for gene replication of COX-1 and COX-2; 3.The presence or absence of a COX-2 inducing agent; 4. COX-2 inducingagents are administered at different concentrations and for differentperiods of time; 5. Duration of incubation with the drug or witharachidonic acid; 6. Variation in the protein concentration in themedium. †Adapted from Pairet, M. and van Ryn, J. (1998) Experimentalmodels used to investigate the differential inhibition ofcyclooxygenase-1 and cyclooxygenase-2 by non-steroidal anti-inflammatorydrugs. Inflamm. Res 47, Supplement 2S93-S101 and incorporated herein byreference.

The enzymes used can be of animal or human origin, they can be native orrecombinant, and they can be used either as purified enzymes, inmicrosomal preparations, or in whole-cell assays. Other system variablesinclude the source of arachidonic acid. PG synthesis can be measuredfrom endogenously released arachidonic acid or exogenously addedarachidonic acid. In the later case, different concentrations are usedin different laboratories.

Second, there are various expression systems for gene replication ofrecombinant COX-1 and COX-2 enzymes. In addition, the cells transfectedwith the Cox-1 or Cox-2 gene can be of diverse origins, for instance,insect cell lines or COS cells. Third, the absence or presence of aCOX-2 inducing agent can vary. Cells that are stably transfected withthe recombinant enzymes express this enzyme constitutively and noinducing agent is used. This is in fundamental contrast with other cellsin which COX-2 has to be induced. Induction of COX-2 is commonlyperformed using bacterial LPS or various cytokines such asinterleukin-1β or tumor necrosis factor. Additionally, these endotoxinsand cytokines are administered at various concentrations.

Fourth, the duration of the incubation with the test agent, the COX-2inducing agent, or with arachidonic acid varies among differentlaboratories. These differences can influence the quantitative outcomeof the study, because the inhibition of COX-2 is time dependent.Finally, the protein concentration of the medium can vary; this is anissue for compounds that can bind avidly to plasma proteins.

A useful assay for COX-2 selectivity would have the followingcharacteristics: (1) whole cells should be used that contain nativehuman enzymes under normal physiological control regarding expression;(2) the cells should also be target cells for the anti-inflammatory andadverse effects of the compounds; (3) COX-2 should be induced, therebysimulating an inflammatory process, rather than being constitutivelyexpressed; and (4) PG synthesis should be measured from arachidonic acidreleased from endogenous stores rather than from exogenously addedarachidonic acid.

Differences in methodology can explain a dramatic difference in theresults obtained for COX inhibition. For example, when assayed againstthe purified enzyme, ursolic acid exhibited an IC₅₀ of 130 μM, faroutside of possible physiologically obtainable concentrations [Ringbom,T. et al. (1998) Ursolic acid from Plantago major, a selective inhibitorof cyclooxygenase-2 catalyzed prostaglandin biosynthesis. J Nat Prod 61,1212-1215]. In the RAW 264.7 murine macrophage line, Suh et al. reportan IC₅₀ for ursolic acid of approximately 40 μM [Suh, N., et al. (1998)Novel triterpenoids suppress inducible nitric oxide synthase (iNOS) andinducible cyclooxygenase (COX-2) in mouse macrophages. Cancer Res 58,717-723]; and in phorbol 12-myristate 13-acetate stimulated humanmammary cells, the approximate median inhibitory concentration ofursolic acid was 3.0 μM [Subbaramaiah, K. et al. (2000) Ursolic acidinhibits cyclooxygenase-2 transcription in human mammary epithelialcells. Cancer Res 60, 2399-2404].

No laboratory has, as yet, developed an ideal assay for COX-2selectivity. The whole cell system most commonly used for Rx and OTCproducts is the human whole blood assay developed by the William HarveyInstitute [Warner, T. D. et al. (1999) Nonsteroid drug selectivities forcyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated withhuman gastrointestinal toxicity: a full in vitro analysis. Proc NatlAcad Sci USA 96, 7563-7568]. To date, this assay format has developedmore data supporting clinical relevance than any other. However, newresearch in the role of constitutive expression of COX-2 in normalgastric mucosa necessitates revisiting the relevance of the use ofplatelets to model COX-1 inhibition in the absence of COX-2. Theextrapolation of gastrotoxicity from platelet studies is no longer on asound molecular basis. The validation of a human gastric mucosal cellline for establishing the potential target tissue toxicity ofcyclooxygenase inhibitors represents a critical need for the developmentof safe and effective anti-inflammatory agents.

NF-κB, a heterodimer of the proteins p50 and RelA, is an inducibleeukaryotic DNA binding protein complex that is broadly expressed andplays a pivotal role in regulating multiple biological responses, suchas the inflammatory and immune responses in mammalian cells. NF-κBregulate the expression of genes encoding cytokines, chemokines,adhesion molecules, and antimicrobial peptides. Targets of NF-κB includeIL-2, the IL-2 receptor, and acute-phase proteins of the liver. Inaddition to its role in immune responses, NF-κB activation overrides theapoptotic response to TNF and Fas, allowing for proliferation instead.

As shown in FIG. 9, NF-κB is cytoplasmic when inactive, maintained thereby IκB. Various stimuli lead to activation of IKK (IκB Kinase), whichphosphorylates IκB, marking it for ubiquitination and degradation. OnceIκB is degraded, NF-κB is freed to initiate transcription. Followingtranscriptional activation of a gene, NF-κB is also rapidly degraded.

Therefore, it would be useful to identify a composition that wouldmodulate expression or activity of NF-κB at the onset of inflammation todecrease the inflammatory response. Additionally, compositions that actas modulators of NF-κB can affect a wide variety of disorders in amammalian body. As a result of inhibiting NFκB, which is a transcriptionfactor for COX-2, the expression of COX-2 can be down-regulated.

An ideal formulation for the treatment of inflammation would inhibit theinduction and activity of COX-2 without inhibiting the synthesis of PGE2in gastric mucosal cells. However, conventional non-steroidalanti-inflammatory drugs lack the specificity of inhibiting COX-2 withoutaffecting gastric PGE2 synthesis and are at risk to cause damages on thegastrointestinal system, when used for extended periods. Indeed, eventhe newly developed, anti-inflammatory drugs such as rofecoxib (Vioxx®,Merck & Co., Inc.) and celexocib (Celebrex®, Pfizer, Inc.) produceuntoward gastric toxicity in the form of induced spontaneous bleedingand delay of gastric ulcer healing.

Thus, it would be useful to identify a natural formulation of compoundsthat would specifically inhibit or prevent the synthesis ofprostaglandins by COX-2 with little or no effect on synthesis of PGE2 inthe gastric mucosa. Such a formulation, which would be useful forpreserving the health of joint tissues, for treating arthritis or otherinflammatory conditions, has not previously been discovered. The term“specific or selective COX-2 inhibitor” was coined to embrace compoundsor mixtures of compounds that selectively inhibit COX-2 over COX-1.However, while the implication is that such a calculated selectivitywill result in lower gastric irritancy, unless the test materials areevaluated in gastric cells, the term “selective COX-2 inhibitor” doesnot carry assurance of safety to gastrointestinal cells. Only testing ofcompound action in target tissues, inflammatory cells and gastricmucosal cells will identify those agents with low potential for stomachirritation.

While glucosamine is generally accepted as being effective and safe fortreating osteoarthritis, medical intervention into the treatment ofdegenerative joint diseases is generally restricted to the alleviationof its acute symptoms. Physicians generally utilize non-steroidal andsteroidal anti-inflammatory drugs for treatment of osteoarthritis. Thesedrugs, however, are not suited for long-term therapy because they notonly lack the ability to protect cartilage, they can actually lead todegeneration of cartilage or reduction of its synthesis. Moreover, mostnon-steroidal, anti-inflammatory drugs damage the gastrointestinalsystem when used for extended periods. Thus, new treatments forarthritis and osteoarthritis combining anti-inflammatory agents withcartilage rebuilding agents are urgently needed.

The joint-protective properties of glucosamine would make it anattractive therapeutic agent for osteoarthritis except for twodrawbacks: (1) the rate of response to glucosamine treatment is slowerthan for treatment with anti-inflammatory drugs, and (2) glucosamine mayfail to fulfill the expectation of degenerative remission. In studiescomparing glucosamine with non-steroidal anti-inflammatory agents, forexample, a double-blinded study comparing 1500 mg glucosamine sulfateper day with 1200 mg ibuprofen, demonstrated that pain scores decreasedfaster during the first two weeks in the ibuprofen patients than in theglucosamine-treated patients. However, the reduction in pain scorescontinued throughout the trial period in patients receiving glucosamineand the difference between the two groups turned significantly in favorof glucosamine by week eight. Lopes Vaz, A., Double-blind clinicalevaluation of the relative efficacy of ibuprofen and glucosaminesulphate in the management of osteoarthritis of the knee in outpatients,8 Curr. Med Res Opin. 145-149 (1982). Thus, glucosamine may relieve thepain and inflammation of arthritis, but at a slower rate than theavailable anti-inflammatory drugs.

An ideal formulation for the normalization of cartilage metabolism ortreatment of osteoarthritis would provide adequate chondroprotectionwith potent anti-inflammatory activity. The optimal dietary supplementfor osteoarthritis should enhance the general joint rebuilding qualitiesoffered by glucosamine and attenuate the inflammatory response withoutintroducing any harmful side effects. It should be inexpensivelymanufactured and comply with all governmental regulations.

However, the currently available glucosamine formulations have not beenformulated to optimally attack and alleviate the underlying causes ofosteoarthritis and rheumatoid arthritis. Moreover, as with manycommercial herbal and dietary supplements, the available formulations donot have a history of usage, nor controlled clinical testing, that mightensure their safety and efficacy.

Therefore, it would be useful to identify a composition that wouldspecifically inhibit or prevent the expression of COX-2 enzymaticactivity in inflammatory cells, while having little or no effect on PGE₂synthesis in gastric mucosal cells so that these formulations could beused with no gastrointestinal upset. Furthermore, such formulationsshould allow for healing of preexisting ulcerative conditions in thestomach.

SUMMARY OF THE INVENTION

Thus, it would be useful to identify a formulation of compounds thatwould modulate an inflammatory response. It would also be useful toidentify a formulation of compounds that would modulate NFκB. Such aformulation has widespread applications.

It would also be useful to identify a formulation of compounds thatwould inhibit expression of COX-2, inhibit prostaglandin synthesisselectively in target cells, or inhibit inflammation responseselectively in target cells. For example, it would also be useful toidentify a formulation of compounds that would specifically inhibit orprevent the synthesis of prostaglandins by COX-2 in inflammatory cellswith little or no effect on PGE₂ synthesis in gastric mucosal cells.Such a formulation, which would be useful for preserving the health ofjoint tissues, for treating arthritis or other inflammatory conditions,has not previously been discovered. Preferably, the formulations have amedian effective concentration for COX-2 inhibition in inflammatorycells that is minimally ten times greater than the median effectiveconcentration for the inhibition of PGE₂ synthesis in gastric cells. Forexample, if the median inhibitory concentration for COX-2 of a testformulation was 0.2 μg/mL in the murine macrophage RAW 264.7, theformulation would not be considered to have low potential for gastricirritancy unless the median inhibitory concentration for PGE₂ synthesisin gastric cells was equal to or greater than 2 μg/mL.

A preferred embodiment comprises compositions containing at least onefraction isolated or derived from hops (Humulus lupulus). Examples offractions isolated or derived from hops are alpha acids, isoalpha acids,reduced isoalpha acids, tetra-hydroisoalpha acids, hexa-hydroisoalphaacids, beta acids, and spent hops. Preferred compounds of fractionsisolated or derived from hops, include, but are not limited to,humulone, cohumulone, adhumulone, isohumulone, isocohumulone,isoadhumulone, dihydro-isohumulone, dihydro-isocohumulone,dihydro-adhumulone, tetrahydro-isohumulone, tetrahydro-isocohumulone,tetrahydro-adhumulone, hexahydro-isohumulone, hexahydro-isocohumulone,and hexahydro-adhumulone. Preferred compounds can also bearsubstituents, such as halogens, ethers, and esters.

Other embodiments relate to combinations of components. One embodimentrelates to compositions that include, as a first component, an activeingredient isolated or derived from an extract of hops and as a secondcomponent at least one member selected from the group consisting ofrosemary (Rosmarinus officinalis L.), an extract or compound derivedfrom rosemary, a triterpene species or derivatives or conjugatesthereof, a diterpene lactone species or derivatives or conjugatesthereof, and tryptanthrin or conjugates thereof. Another embodimentrelates to compositions that include, as a first component, tryptanthrinor conjugates thereof and as a second component at least one memberselected from the group consisting of an active ingredient isolated orderived from an extract of hops, rosemary, an extract or compoundderived from rosemary, a triterpene species or derivatives or conjugatesthereof, and a diterpene lactone species or derivatives or conjugatesthereof. As used herein, an extract refers to an extract containing anactive ingredient that effects an activity, for example, inhibitinginflammation, inhibiting inducibility or activity of COX-2, inhibitingprostaglandin synthesis, modulating NFκB, and the like.

Preferred compositions can inhibit the inducibility or activity ofCOX-2. Compositions of the invention can also function to modulate NFκB.Preferred compositions also can inhibit prostaglandin synthesisselectively in target cells. Preferred compositions also can inhibitinflammation response selectively in target cells. As used herein, anextract refers to an extract containing an active ingredient thateffects an activity, for example, inhibiting inflammation, inhibitinginducibility or activity of COX-2, inhibiting prostaglandin synthesis,modulating NFκB, and the like.

The compositions have widespread applications. Preferred compositionscan be useful for treating conditions, such as cancer, autoimmunediseases, inflammatory diseases, or neurological diseases, and obesity.Preferred compositions are also believed to be useful for treatingconditions, such as HIV-1 infections, rhinovirus infections, andcardiovascular diseases.

Preferred compositions would be useful for, but not limited to, thetreatment of inflammation in a subject, and for treatment of otherinflammation-associated disorders, such as an analgesic in the treatmentof pain and headaches, or as an antipyretic for the treatment of fever.Preferred compositions would be useful to treat arthritis, including butnot limited to rheumatoid arthritis, spondyloathopathies, goutyarthritis, osteoarthritis, systemic lupus erythematosis, and juvenilearthritis.

Preferred compositions would be useful in the treatment of asthma,bronchitis, menstrual cramps, tendonitis, bursitis, and skin-relatedconditions such as psoriasis, eczema, burns and dermatitis. Preferredcompositions also would be useful to treat gastrointestinal conditionssuch as inflammatory bowel disease, Crohn's disease, gastritis,irritable bowel syndrome and ulcerative colitis and for the preventionor treatment of cancer such as colorectal cancer.

Further, preferred compositions would be useful in treating inflammationin such diseases as vascular diseases, migraine headaches, periarteritisnodosa, thyroiditis, aplastic anemia, Hodgkin's disease, sclerodma,rheumatic fever, type I diabetes, myasthenia gravis, multiple sclerosis,sacoidosis, nephrotic syndrome, Behchet's syndrome, polymyositis,gingivitis, hypersensitivity, swelling occurring after injury,myocardial ischemia, peridontal disease, fibromyalgia, atopicdermatitis, insulitis and the like.

Additionally, preferred compositions would also be useful in thetreatment of ophthalmic diseases, such as retinopathies, conjunctivitis,uveitis, ocular photophobia, and of acute injury to the eye tissue.Preferred compositions would also be useful in the treatment ofpulmonary inflammation, such as that associated with viral infectionsand cystic fibrosis.

Preferred compositions would also be useful for the treatment of certainnervous system disorders such as cortical dementias includingAlzheimer's disease. As inhibitors of COX-2 mediated biosynthesis ofPGE₂ in inflammatory cells, these compositions would also be useful inthe treatment of allergic rhinitis, respiratory distress syndrome,endotoxin shock syndrome, atherosclerosis, and central nervous systemdamage resulting from stroke, ischemia and trauma.

Preferred embodiments further provides a composition to increase therate at which glucosamine or chondrotin sulfate function to normalizejoint movement or reduce the symptoms of osteoarthritis.

Preferred embodiments also provide for methods of identifyingcompositions that would specifically inhibit or prevent the synthesis ofprostaglandins by COX-2 in inflammatory cells with little or no effecton PGE₂ synthesis in gastric mucosal cells.

In addition, the compositions of the invention would also be useful forthe treatment of obesity, syndrome X, and the like, by reversinghyperglycemia, hyperinsulinemia, and dyslipidermia by normalizingactivation of IkkB kinase beta and hence properly modulating its effecton transcription of NFκB-activated genes, effectively breaking thecytokine (for example, tumor necrosis factor alpha (TNFα) feed-back loopleading to these conditions and diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the induction of cyclooxygenase-2 and the metabolism ofarachidonic acid to prostaglandins and other eicosanoids by thecyclooxygenase enzymes. The action of non-steroidal anti-inflammatoryagents is through direct inhibition of the cyclooxygenase enzymes.

FIG. 2 shows an outline of fractions and compounds that can be obtainedfrom hops.

FIG. 3 illustrates [A] the alpha-acid genus (AA) and representativespecies humulone (R═—CH₂CH(CH₃)₂), cohumulone (R═, —CH(CH₃)₂), andadhumulone (R═—CH(CH₃)CH₂CH₃); [B] the isoalpha acid genus (IAA) andrepresentative species isohumulone (R═—CH₂CH(CH₃)₂), isocohumulone (R═,—CH(CH₃)₂), and isoadhumulone (R═—CH(CH₃)CH₂CH₃); [C] the reducedisomerized isoalpha acid genus (RIAA) and representative speciesdihydro-isohumulone (R═—CH₂CH(CH₃)₂) dihydro-isocohumulone (R═,—CH(CH₃)₂), and dihydro-adhumulone (R═—CH(CH₃)CH₂CH₃); [D] thetetra-hydroisoalpha acid genus (THIAA) and representative speciestetra-hydro-isohumulone (R═—CH₂CH(CH₃)₂), tetra-hydro-isocohumulone((R═, —CH(CH₃)₂), and tetra-hydro-adhumulone R═—CH(CH₃)CH₂CH₃); [E] andthe hexa-hydroisoalpha acid (HHIAA) genus with representative specieshexa-hydro-isohumulone (R═—CH₂CH(CH₃)₂) hexa-hydro-isocohumulone (R═,—CH(CH₃)₂), and hexa-hydro-adhumulone (R═—CH(CH₃)CH₂CH₃).

FIG. 4 illustrates the chemical structure of tryptanthrin.

FIG. 5 illustrates the general chemical structures of the triterpenegenus [A] and ursolic acid [B] and oleanolic acid [C] as a specieswithin that genus.

FIG. 6 [A] shows representative immunoblots demonstrating constitutiveCOX-1 and COX-2 expression in AGS human gastric mucosal cells. The AGShuman gastric cell line was cultured in 6-well plates at 37° C. with 5%CO₂ in a humidified incubator for 24 hours. Cells were lysed on ice inlysis buffer and protein concentration determined. Fifty μg of celllysate were solubilized, fractionated on a 10% polyacrylamide gelcontaining sodium dodecylsulfate (SDS), and transferred onto anitrocellulose membrane. The membranes were incubated in a blockingbuffer and then incubated with the respective primary antibody for 1 hat room temperature. Following primary antibody incubation, the blotswere washed three times with Tris-buffered saline and then incubatedwith the secondary antibody for 1 h. Protein bands were visualized usingenhanced chemiluminescence. FIG. 6 [B] shows densitometric analysis ofthe immunoblots shown in FIG. 6A. Band intensities were evaluatedthrough densitometric analysis, computed using ScanAnalysis® software(BIOSOFT, Ferguson, Mo.), and recorded as arbitrary Density Units (DU).

FIG. 7 [A] shows the percent inhibition of PGE₂ synthesis inLPS-stimulated RAW 264.7 cells by plasma samples from a human volunteerreceiving 880 mg t.i.d. of a test hops derivative formulation. Whitebars are means of raw data and dark bars are those means computed withthe elimination of outliers (never more than two of the eightreplicates). The gel capsules of the test formulation contained 200 mgreduced isomerized alpha-acids, 200 mg rosemary extract and 40 mgoleanolic acid. FIG. 7[B] is an estimate of the plasma concentrations oftest material at each post-dosing time capable of inhibiting PGE₂synthesis in LPS-stimulated RAW 264.7 cells assuming a constant 5:5:1ratio of components.

FIG. 8 illustrates the induction of PGE₂ synthesis by mite allergen inA549 pulmonary cells treated for 24 hours.

FIG. 9 shows a pathway of activation of NF-κB. In the cytoplasm, NF-κBis inhibited by Iκb. An upstream activating signal may causephosphorylation of IκB by IKK (IκB kinase). This triggers thedegradation of IκB through the ubiquitin system. Once freed from IκB,the free NF-κB can then translocate to the nucleus and activatetranscription.

FIG. 10 shows dose-related inhibition of PGE₂ biosynthesis for RIAA(shaded bar) and IAA (white bar) treated RAW 264.7 cells followingovernight. LPS-stimulation prior to the addition of test material.

FIG. 11 shows comparison of log IC₅₀ ratios and ranking of potentialgastropathy. Log IC₅₀ ratios using the William Harvey Modified Assay(WHMA) are expressed WHMA COX-1/WHMA COX-2 from Warner, et al. (whitebars) and Mitchell, et al. (dark bars) are shown in [A} with log IC₅₀ratios (AGS/WHMA COX-2) for AGS cells treated with A23187 [B], 100 μMarachidonic acid [C], or 5 μM arachidonic acid [D]. Values to the rightof 0 indicate decreasing probability of gastropathy, whereas values tothe left of 0 indicate increasing probability of gastropathy.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention relates to the discovery that that a supragenus ofcomponents isolated or derived from hops and other compounds result intissue-specific or cell-specific inhibition of COX-2 expression.Importantly, these compounds are not believed to directly inhibit COX-2or other enzymes within the prostaglandin synthesis pathway. Preferredembodiments provide compositions and methods for inhibiting COX-2expression, inhibiting prostanglandin synthesis selectively in targettissues or cells, or inhibiting inflammation response selectively intarget tissues or cells. Compositions and methods of the invention canalso modulate NFκB.

A preferred embodiment comprises compositions containing fractions orcompounds isolated or derived from hops. Examples of fractions isolatedor derived from hops are alpha acids, isoalpha acids, reduced isoalphaacids, tetra-hydroisoalpha acids, hexa-hydroisoalpha acids, beta acids,and spent hops. Preferred compounds of the fractions isolated or derivedfrom hops can be represented by a supragenus below:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; wherein R″ is selected from the groupconsisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; and wherein R,T, X, and Z are independently selected from the group consisting of H,F, Cl, Br, I, and π orbital, with the proviso that if one of R, T, X, orZ is a π orbital, then the adjacent R, T, X, or Z is also a π orbital,thereby forming a double bond.

Other preferred compounds of the fractions isolated or derived from hopscan be represented by a genus below:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; and wherein R″ is selected from thegroup consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

Other preferred compounds of the fractions isolated or derived from hopscan be represented by a genus below:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; and wherein R″ is selected from thegroup consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

Examples of preferred compounds of an ingredient isolated or derivedfrom hops, include, but are not limited to, humulone, cohumulone,adhumulone, isohumulone, isocohumulone, isoadhumulone,dihydro-isohumulone, dihydro-isocohumulone, dihydro-adhumulone,tetrahydro-isohumulone, tetrahydro-isocohumulone, tetrahydro-adhumulone,hexahydro-isohumulone, hexahydro-isocohumulone, andhexahydro-adhumulone. The preferred compounds can bear substituents, asshown in the formula above.

Another embodiment comprises composition containing tryptanthrin andconjugates thereof.

Other embodiments relate to combinations of components. In particularembodiments, the compositions of the invention can function tospecifically inhibit COX-2 expression, to modulate NFκB, to inhibitprostaglandin synthesis selectively in target cells, or to inhibitinflammation response selectively in target cells. The compositions canexhibit synergistic activity.

One embodiment relates to compositions that include, as a firstcomponent, an active ingredient isolated or derived from an extract ofhops and as a second component at least one member selected from thegroup consisting of rosemary, an extract or compound derived fromrosemary, a triterpene species or derivatives or conjugates thereof, aditerpene lactone species or derivatives or conjugates thereof, andtryptanthrin or conjugates thereof. Another embodiment relates tocompositions that include, as a first component, tryptanthrin orconjugates thereof and as a second component at least one memberselected from the group consisting of an active ingredient isolated orderived from an extract of hops, rosemary, an extract or compoundderived from rosemary, a triterpene species or derivatives or conjugatesthereof, and a diterpene lactone species or derivatives or conjugatesthereof.

As used herein, the term “dietary supplement” refers to compositionsconsumed to affect structural or functional changes in physiology. Theterm “therapeutic composition” refers to any compounds administered totreat or prevent a disease.

As used herein, the term “effective amount” means an amount necessary toachieve a selected result. Such an amount can be readily determinedwithout undue experimentation by a person of ordinary skill in the art.

As used herein, the term “substantial” means being largely but notwholly that which is specified.

As used herein, the term “COX inhibitor” refers to a composition ofcompounds that is capable of inhibiting the activity or expression ofCOX-2 enzymes or is capable of inhibiting or reducing the severity,including pain and swelling, of a severe inflammatory response.

As used herein, the terms “derivatives” or a matter “derived” refer to achemical substance related structurally to another substance andtheoretically obtainable from it, i.e. a substance that can be made fromanother substance. Derivatives can include compounds obtained via achemical reaction.

As used herein, the term “inflammatory cell” refers to those cellularmembers of the immune system, for example B and T lymphocytes,neutrophils or macrophages involved in synthesis of prostaglandins inresponse to inflammatory signals such as interleukins, tumor necrosisfactor, bradykinin, histamine or bacterial-derived components.

As used herein, the term “target cells” refers to that cell populationin which the inhibition of PGE₂ or other prostaglandin synthesis isdesired, such as inflammatory cells, tumor cells, or pulmonary cells.Alternatively, “non-target cells” refers to that cell population inwhich the inhibition of PGE₂ or other prostaglandin synthesis is notdesired, such as the gastric mucosal, neural or renal cells.

As used herein, the term “hop extract” refers to the solid materialresulting from (1) exposing a hops plant product to a solvent, (2)separating the solvent from the hops plant products, and (3) eliminatingthe solvent.

As used herein, the term “solvent” refers to a liquid of aqueous ororganic nature possessing the necessary characteristics to extract solidmaterial from the hop plant product. Examples of solvents would include,but not limited to, water, steam, superheated water, methanol, ethanol,hexane, chloroform, liquid CO₂, liquid N₂ or any combinations of suchmaterials.

As used herein, the term “CO₂ extract” refers to the solid materialresulting from exposing a hops plant product to a liquid orsupercritical CO₂ preparation followed by removing the CO₂.

As used herein, the term “spent hops” refers to the solid andhydrophilic residue from extract of hops.

As used herein, the term “alpha acid” refers to compounds refers tocompounds collectively known as humulones and can be isolated from hopsplant products including, among others, humulone, cohumulone,adhumulone, hulupone, and isoprehumulone.

As used herein, the term “isoalpha acid” refers to compounds isolatedfrom hops plant products and subsequently have been isomerized. Theisomerization of alpha acids can occur thermally, such as boiling.Examples of isoalpha acids include, but are not limited to, isohumulone,isocohumulone, and isoadhumulone.

As used herein, the term “reduced isoalpha acid” refers to alpha acidsisolated from hops plant product and subsequently have been isomerizedand reduced, including cis and trans forms. Examples of reduced isoalphaacids (RIAA) include, but are not limited to, dihydro-isohumulone,dihydro-isocohumulone, and dihydro-adhumulone.

As used herein, the term “tetra-hydroisoalpha acid” refers to a certainclass of reduced isoalpha acid. Examples of tetra-hydroisoalpha acid(THIAA) include, but are not limited to, tetra-hydro-isohumulone,tetra-hydro-isocohumulone and tetra-hydro-adhumulone.

As used herein, the term “hexa-hydroisoalpha acid” refers to a certainclass of reduced isoalpha acid. Examples of hexa-hydroisoalpha acids(HHIAA) include, but are not limited to, hexa-hydro-isohumulone,hexa-hydro-isocohumulone and hexa-hydro-adhumulone.

As used herein, the term “beta-acid fraction” refers to compoundscollectively known as lupulones including, among others, lupulone,colupulone, adlupulone, tetrahydroisohumulone, and hexahydrocolupulone.

As used herein, the term “essential oil fraction” refers to a complexmixture of components including, among others, myrcene, humulene,beta-caryophyleen, undecane-2-on, and 2-methyl-but-3-en-ol.

As used herein, “conjugates” of compounds means compounds covalentlybound or conjugated to a member selected from the group consisting ofmono- or di-saccharides, amino acids, sulfates, succinate, acetate, andglutathione. Preferably, the mono- or di-saccharide is a member selectedfrom the group consisting of glucose, mannose, ribose, galactose,rhamnose, arabinose, maltose, and fructose.

As used herein, the term “fats” refers to triacylglyerol esters of fattyacids.

As used herein, the term “waxes” refers to triacylglycerol ethers of oresters of extremely long chain (>25 carbons) fatty alcohols or acids.

Hops

Hop extraction in one form or another goes back over 150 years to theearly nineteenth century when extraction in water and ethanol was firstattempted. Even today an ethanol extract is available in Europe, but byfar the predominant extracts are organic solvent extracts (hexane) andCO₂ extracts (supercritical and liquid). CO₂ (typically at 60 barspressure and 50 to 10° C.) is in a liquid state and is a relativelymild, non-polar solvent highly specific for hop soft resins and oils.Beyond the critical point, typically at 300 bars pressure and 60° C.,CO₂ has the properties of both a gas and a liquid and is a much strongersolvent. The composition of the various extracts is compared in Table 2.

TABLE 2 Hop Extracts (Percent W/W) Organic Component Hops SolventSuper-Critical CO₂ Liquid CO₂ Total resins 12-20 15-60 75-90 70-95Alpha-acids  2-12  8-45 27-55 30-60 Beta-acids  2-10  8-20 23-33 15-45Essential oils 0.5-1.5 0-5 1-5  2-10 Hard resins 2-4  2-10  5-11 NoneTannins  4-10 0.5-5   0.1-5   None Waxes 1-5  1-20  4-13  0-10 Water 8-12  1-15 1-7 1-5

At its simplest, hop extraction involves milling, pelleting andre-milling the hops to spread the lupulin, passing a solvent through apacked column to collect the resin components and finally, removal ofthe solvent to yield a whole or “pure” resin extract.

The main organic extractants are strong solvents and in addition tovirtually all the lupulin components, they extract plant pigments,cuticular waxes, water and water-soluble materials.

Supercritical CO₂ is more selective than the organic solvents andextracts less of the tannins and waxes and less water and hencewater-soluble components. It does extract some of the plant pigmentslike chlorophyll but rather less than the organic solvents do. LiquidCO₂ is the most selective solvent used commercially for hops and henceproduces the most pure whole resin and oil extract. It extracts hardlythe hard resins or tannins, much lower levels of plant waxes, no plantpigments and less water and water-soluble materials.

As a consequence of this selectivity and the milder solvent properties,the absolute yield of liquid CO₂, extract per unit weight of hops isless than when using the other mentioned solvents. Additionally, theyield of alpha acids with liquid CO₂ (89-93%) is lower than that ofsupercritical CO₂ (91-94%) or the organic solvents (93-96%). Followingextraction there is the process of solvent removal, which for organicsolvents involves heating to cause volatilization. Despite this, traceamounts of solvent do remain in the extract. The removal of CO₂,however, simply involves a release of pressure to volatize the CO₂.

As shown in FIG. 2, hops CO₂ extracts can be fractionated intocomponents, including hops oils, beta acids, and alpha acids. Hops oilsinclude, but not limited to, humulene, beta-caryophyllene, mycrene,farnescene, gamma-cadinene, alpha-selinene, and alpha-cadinene. Betaacids include, but are not limited to, lupulone, colupulone, adlupulone,tetrahydroisohumulone, and hexahydrocolupulone, collectively known aslupulones. Beta acids can be isomerized and reduced. Beta acids arereduced to give tetra-beta acids. Alpha acids include, but are notlimited to, humulone, cohumulone, adhumulone, hulupone, andisoprehumulone. Alpha acids can be isomerized to give isoalpha acids.Iso-alpha acids can be reduced to give reduced-isoalpha acids,tetra-hydroisoalpha acids, and hexa-hydroisoalpha acids.

A preferred embodiment comprises compositions containing fractions orcompounds isolated or derived from hops. Examples of fractions isolatedor derived from hops are alpha acids, isoalpha acids, reduced isoalphaacids, tetra-hydroisoalpha acids, hexa-hydroisoalpha acids, beta acids,and spent hops. Preferred compounds of the fractions isolated or derivedfrom hops can be represented by a supragenus below:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; wherein R″ is selected from the groupconsisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; and wherein R,T, X, and Z are independently selected from the group consisting of H,F, Cl, Br, I, and π orbital, with the proviso that if one of R, T, X, orZ is a π orbital, then the adjacent R, T, X, or Z is also a π orbital,thereby forming a double bond.

Other preferred compounds of the fractions isolated or derived from hopscan be represented by a genus below:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; and wherein R″ is selected from thegroup consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

Other preferred compounds of the fractions isolated or derived from hopscan be represented by a genus below:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; and wherein R″ is selected from thegroup consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

As shown in FIG. 3, examples of preferred compounds of an ingredientisolated or derived from hops, include, but are not limited to,humulone, cohumulone, adhumulone, isohumulone, isocohumulone,isoadhumulone, dihydro-isohumulone, dihydro-isocohumulone,dihydro-adhumulone, tetrahydro-isohumulone, tetrahydro-isocohumulone,tetrahydro-adhumulone, hexahydro-isohumulone, hexahydro-isocohumulone,and hexahydro-adhumulone. The preferred compounds can bear substituents,as shown in the formula above.

The identification of humulone from hops extract as an inhibitor of boneresorption is reported in Tobe, H. et al. 1997. (Bone resorptionInhibitors from hop extract. Biosci. Biotech. Biochem 61(1)158-159.)Tobe et al. merely discloses the use of humulone, cohumulone,adhumulone, isohumulone, isocohumulone, and isoadhumulone for treatingosteoporosis. Later studies by the same group characterized themechanism of action of humulone as inhibition of COX-2 genetranscription following TNFalpha stimulation of MC3T3, E1 cells[Yamamoto, K. 2000. Suppression of cyclooxygenase-2 gene transcriptionby humulon of beer hop extract studied with reference to theglucocorticoid receptor. FEBS Letters 465:103-106]. The authorsconcluded that the action of humulone (also humulon) was similar to thatof glucocorticoids, but that humulone did not function through theglucocorticoid receptor. While these results establish that humuloneinhibits PGE₂ synthesis in MC3T3 cells (osteoblasts) at the gene level,one skilled in the art would not assume that these results wouldnecessarily occur in immune inflammatory cells or other cell lines.Example 5 herein demonstrates the high degree of tissue selectivity ofhops compounds and derivatives.

Preferred embodiments provide compositions and methods for inhibitingexpression of COX-2, modulating NFκB tissue specifically and cellspecifically, inhibiting synthesis of prostaglandins selectively intarget cells, and inhibiting inflammatory response selectively in targetcells. Preferred methods comprise a step of administering to a mammal acomposition of the preferred embodiments. Preferred embodiments comprisea fraction isolated or derived from hops. A certain compositioncomprises alpha acids, isoalpha acids, reduced isoalpha acids,tetra-hydroisoalpha acids, hexa-hydroisoalpha acids, beta acids, orspent hops from hops extract or derivatives thereof. Preferred compoundsof the fractions isolated or derived from hops can be represented by asupragenus below:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; wherein R″ is selected from the groupconsisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; and wherein R,T, X, and Z are independently selected from the group consisting of H,F, Cl, Br, I and π orbital, with the proviso that if one of R, T, X, orZ is a π orbital, then the adjacent R, T, X, or Z is also a π orbital,thereby forming a double bond. Other preferred compounds of thefractions isolated or derived from hops can be represented by a genusbelow:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; and wherein R″ is selected from thegroup consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃. Otherpreferred compounds of the fractions isolated or derived from hops canbe represented by a genus below:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; and wherein R″ is selected from thegroup consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃. Thepreferred embodiments contemplate compositions comprising beta acids orisomerized or reduced beta acids. Preferably, the alpha acid, isoalphaacid, reduced isoalpha acid, tetra-hydroisoalpha acid,hexa-hydroisoalpha acid, beta acid, or spent hops of the preferredembodiments is made from hops extract. More preferably, the alpha acid,isoalpha acid, reduced isoalpha acid, tetra-hydroisoalpha acid,hexa-hydroisoalpha acid, beta acid, or spent hops of the preferredembodiments is made from CO₂ extract of hops.Tryptanthrin

Preferred embodiments can provide compositions and methods forinhibiting expression of COX-2, modulating NFκB tissue specifically andcell specifically inhibiting synthesis of prostaglandins selectively intarget cells, and inhibiting inflammatory response selectively in targetcells. Preferred methods comprise a step of administering to a mammal acomposition of the preferred embodiments. A certain compositioncomprises tryptanthrin and conjugates thereof.

Depicted in FIG. 4, tryptanthrin is a natural compound found in certainherbs, such as Polygonum tinctorium and Isatis tinctoria. In traditionalChinese medicine this herb is known as Da Qing Ye or Qing Dai. The herbhas demonstrated antibacterial and antiviral activity. It hasantipyretic, anti-inflammatory and choleretic properties. Increasedphagocytic activity of leukocytes and relaxation of intestinal smoothmuscle are additional properties of Qing Dai.

Rosemary

Certain of preferred embodiments also include delivering an effectiveamount of rosemary, rosemary extract, or compounds derived from rosemarywith the fraction isolated or derived from hops or tryptanthrin.Preferred additions include, but are not limited to, rosemary, rosemaryextract, or those compounds known to be found in rosemary or extracts ofrosemary. These include 1,8-cineole, 19-alpha-hydroxyursolic acid,2-β-hydroxyoleanolic acid, 3-O-acetyloleanolic acid, 3-O-acetylursolicacid, 6-methoxy-luteolin-7-glucoside, 6-methoxyluteolin,6-methoxyluteolin-7-glucoside, methoxyluteolin-7-methylether,7-ethoxy-rosmanol, 7-methoxy-rosmanol, alpha-amyrin, alpha-humulene,alpha-hydroxyhydrocaffeic acid, alpha-pinene, alpha-terpinene,alpha-terpinenyl acetate, alpha-terpineol, alpha-thujone, apigenin,apigenin-7-glucoside, curcumene, benzyl-alcohol, β-amyrenone, β-amyrin,β-elemene, β-pinene, betulin**, betulinic acid**, borneol,bornyl-acetate, caffeic acid, camphene, camphor, carnosic acid**,carnosol**, carvacrol**, carvone, caryophyllene, caryophyllene-oxide,chlorogenic acid**, diosmetin**, gamma-terpinene, hesperidin,isoborneol, limonene*, luteolin*,luteolin-3′-O-(3″-O-acetyl)-β-D-glucuronide,luteolin-3′-O-(4″-O-acetyl)-β-D-glucuronide,luteolin-3′-O-β-D-glucuronide, luteolin-7-glucoside, methyl-eugenol,myrcene, neo-chlorogenic acid, nepetin, octanoic acid, oleanolic acid,p-cymene, piperitenone, rosmanol, rosmaric acid, rosmaricine,rosmaridiphenol, rosemarinic acid, rosmarinol, rosmariquinone, sabinene,sabinyl acetate, salicylates, salicylic acid-2-β-D-glucoside, squalene,terpinen-4-ol, terpinolene, thymol, trans-anethole, trans-carveol,ursolic acid, verbenone, and zingiberene. Of the species listed, thosecontaining at least one asterisk (*) are preferred and those containingtwo asterisks (**) are particularly preferred.

Triterpenes and Diterpene Lactones

Certain of preferred embodiments also include delivering an effectiveamount of a triterpene species or diterpene lactone species with thefraction isolated or derived from hops or tryptanthrin. Preferredtriterpenes include oleanolic acid, and ursolic acid. Both ursolic andoleanolic acid are found in a wide variety of botanicals. Diterpenelactones, such as andrographolide, can be obtained from Andrographispaniculata.

Diterpene lactone species, such as andrographolide, and triterpenes,such as ursolic acid and oleanolic acid, are commonly found in plantsand are used for their anti-inflammatory properties. Theanti-inflammatory effects of these compounds have been described in theliterature since 1960. Their mechanism of action is believed to be due(i) to the inhibition of histamine release from mast cells or (ii) tothe inhibition of lipoxygenase and cyclooxygenase activity therebyreducing the synthesis of inflammatory factors produced during thearachidonic acid cascade. Since andrographolide and oleanolic acid havebeen found to promote the healing of stomach ulcers, it is unlikely thatthe cyclooxygenase activity that is inhibited is COX-1. Also,andrographolide and oleanolic acid are potent antioxidants, capable ofinhibiting the generation of reactive oxygen intermediates and restoringtissue glutathione levels following stress.

For example, botanical sources for ursolic acid can be selected from thegroup consisting of Adina piluifera, Agrimonia eupatoria, Arbutus unedo,Arctostaphylos uva-ursi, Artocarpus heterophyllus, Catalpa bignoniodes,Catharanthus roseus, Chimaphila umbellata, Cornus florida, Cornusofficinalis, Crataegus cuneata, Crataegus laevigata, Crataeguspinnatifida, Cryptostegia grandifolia, Elaeagnus pungens, Eriobotryajaponica, Eucalyptus citriodora, Forsythia suspensa, Gaultheriafragrantissima, Glechoma hederacea, Hedyotis diffusa, Helichrysumangustifolium, Humulus lupulus, Hyssopus officinalis, Ilexparaguariensis, Lavandula angustifolia, Lavandula latifolia, Leonuruscardiaca, Ligustrum japonicum, Limonia acidissima, Lycopus europeus,Malus domestica, Marubium vulgare, Melaleuca leucadendra, Melissaofficinalis, Mentha spicata, Mentha×rotundifolia, Monarda didyma, Neriumoleander, Ocimum basilicum, Ocimum basilicum, Ocimum basilicum, Ocimumbaslicum, Ocimum canum, Origanum majorana, Origanum vulgare, Plantagoasiatica, Plantago major, Plectranthus amboinicus, Prunell vulgaris,Prunella vulgaris, Prunus cerasus, Prunus laurocerasus, Prunus persica,Prunus serotina spp serotina, Psidium guajava, Punica granatum, Pyruscommunis, Rhododendron dauricum, Rhododendron ferrugineum, Rhododendronponticum, Rosmarinus officinalis, Rubus fruticosus, Salvia officinalis,Salvia sclarea, Salvia triloba, Sambucus nigra, Sanguisorba officinalis,Satureja hortensis, Satureja montana, Sorbus aucubaria, Syringavulgaris, Teucrium chamaedrys Teucrium polium, Teucrium spp, Thevetiaperuviana, Thymus serpyllum, Thymus vulgaris, Uncaria tomentosa,Vaccinium corymobosum, Vaccinium myrtillus, Vaccinium vitis idaea,Verbena officinalis, Viburnum opulus var. opulus, Viburnum prunifolium,Vinca minor and Zizyphus jujuba.

Similarly, oleanolic acid is found in Achyranthes aspera, Achyranthesbidentiata, Adina piluifera, Ajpocynum cannabinum, Akebia quinata,Allium cepa, Allium sativum, Arctostaphylos uva-ursi, Calendulaofficinalis, Catharanthus roseus, Centaurium erythraea, Chenopodiumalbum, Citrullus colocynthis, Cnicus benedictus, Cornus officinalis,Crataegus pinnatifida Cyperus rotundus, Daemonorops draco, Diospyroskaki, Elaeagnus pungens, Eleutherococcus senticosus, Eriobotryajaponica, Eugenia caryophyllata, Forsythia suspensa, Glechoma hederacea,Harpagophtum procumbens, Hedera helix, Hedyotis diffusa, Helianthusannuus, Hemsleys amabilis, Humulus lupulus, Hyssopus officinalis, Ilexrotunda, Lavandula latifolia, Leonurus cardiaca, Ligustrum japonicum,Ligustrum lucidum, Liquidambar orientalis, Liquidambar styraciflua,Loranthus parasiticus, Luffa aegyptiaca, Melaleuca leucadendra, Melissaofficinalis, Mentha spicata, Mentha×rotundifolia, Momordicacochinchinensis, Myristica fragrans, Myroxylon balsamum, Neriumoleander, Ocimum suave, Ociumum basilicum, Olea europaea, Origanummajorana, Origanum vulgare, Paederia scandens, Panax ginseng, Panaxjaponicus, Panax quinquefolius, Patrinia scabiosaefolia, Phytolaccaamericana, Plantago major, Plectranthus amboinicus, Prunella vulgaris,Prunus cerasus, Psidium guajava, Pulsatilla chinenisis, Quisqualisindica, Rosmarinus officinalis, Salvaia officinalis, Salvia sclarea,Salvia triloba, Sambucus nigra, Satureja hortensis, Satureja montana,Swertia chinensis, Swertia diluta, Swertia mileensis, Syzygiumaromaticum, Thymus serpyllum, Thymus vulgaris, Trachycarpus fortunei,Uncaria tomentosa, Vaccinium corymbosum, Vaccinium myrtillus, Viburnumprunifolium, Viscum album, Vitis vinifera, or Zizyphus jujuba.

The preferred botanical sources for ursolic acid is a member selectedfrom the group consisting of Ligustrum japonicum, Plantago asiatica,Plantago major, Prunus species, Uncaria tomentosa, Zizyphus jujuba,Cornus officinalis, Eucalyptus citriodora, Forsythia suspensa, Lavandulalatifolia, Malus domestica, Nerium oleander, Ocimum baslicum, Punicagranatum, Pyrus communis, Rosmarinus officinalis, Salvia triloba, Sorbusaucubaria, Vaccinium myrtillus, Vaccinium vitis-idaea, and Viburnumopulus var. opulus. The most preferred botanical sources for ursolicacid is a member selected from the group consisting of Ligustrumjaponicum, Plantago asiatica, Plantago major, Prunus species, Uncariatomentosa, and Zizyphus jujuba.

The preferred botanical source for oleanolic acid is a member selectedfrom the group consisting of Eleutherococcus senticosus, Ligustrumjaponicum, Ligustrum lucidum, Panax ginseng, Panax japonicus, Panaxquinquefolius, Plantago major, Prunella vulgaris, Vitis vinifera,Zizyphus jujuba, Achyranthes bidentiata, Allium cepa, Allium sativum,Cornus officinalis, Daemonorops draco, Forsythia suspensa, Prunuscerasus, Quisqualis indica, Rosmarinus officinalis, Salvia triloba,Syzygium aromaticum, Thymus vulgaris, Uncaria tomentosa, Vacciniumcorymbosum, and Vaccinium myrtillus. The most preferred botanical sourcefor oleanolic acid is a member selected from the group consisting ofEleutherococcus senticosus, Ligustrum japonicum, Ligustrum lucidum,Panax ginseng, Panax japonicus, Panax quinquefolius, Plantago major,Prunella vulgaris Vitis vinifera and Zizyphus jujuba.

FIG. 5 illustrates the general chemical structures of the triterpenegenus and ursolic acid and oleanolic acid as a species within thatgenus. Representative terpenoids within the genus are18-a-glycyrrhetinic acid**, 18-β-glycyrrhetinic acid**,2-a-3-a-dihydrooxyurs-12-3n-28-onic acid*, 3-a-hydroxyursolic acid*,3-oxo-ursolic acid*, betulin**, betulinic acid**, celastrol*, eburicoicacid, friedelin*, glycyrrhizin, gypsogenin, oleanolic acid**, oleanolicacid-3-acetate, pachymic acid, pinicolic acid, sophoradiol,soyasapogenol A, soyasapogenol B, tripterin**, triptophenolide*,tumulosic acid, ursolic acid**, ursolic acid-3-acetate, uvaol*, andβ-sitosterol. Of the species listed, those containing at least oneasterisk (*) are preferred and those containing two asterisks (**) areparticularly preferred.

Examples of diterpene lactone species include, but is not limited to,andrographolide, dehydroandrographolide, deoxyandrographolide,neoandrographolide, selenoandrographolide, homoandrographolide,andrographan, amdrographon, andrographosterin,14-deoxy-11-oxoandrographolide, 14-deoxy-11,12-didehydroandrographolide,andrographiside, and edelin lactone.

Compositions and Synergistic Combinations

Preferred compositions can function to specifically inhibit COX-2expression, to modulate NFκB, to inhibit prostaglandin synthesisselectively in target cells, or to inhibit inflammation responseselectively in target cells. Preferred embodiments include compositionscontaining fractions or compounds isolated or derived from hops orcompositions containing tryptanthrin and conjugates thereof.

A preferred embodiment comprises compositions containing fractions orcompounds isolated or derived from hops. Examples of fractions isolatedor derived from hops are alpha acids, isoalpha acids, reduced isoalphaacids, tetra-hydroisoalpha acids, hexa-hydroisoalpha acids, beta acids,and spent hops. Preferred compounds of the fractions isolated or derivedfrom hops can be represented by a supragenus below:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; wherein R″ is selected from the groupconsisting of CH(CH₃), CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; and wherein R, T,X, and Z are independently selected from the group consisting of H, F,Cl, Br, I and π orbital, with the proviso that if one of R, T, X, or Zis a π orbital, then the adjacent R, T, X, or Z is also a π orbital,thereby forming a double bond.

Other preferred compounds of the fractions isolated or derived from hopscan be represented by a genus below:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; and wherein R″ is selected from thegroup consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

Other preferred compounds of the fractions isolated or derived from hopscan be represented by a genus below:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; and wherein R″ is selected from thegroup consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

Examples of preferred compounds of an ingredient isolated or derivedfrom hops, include, but are not limited to, humulone, cohumulone,adhumulone, isohumulone, isocohumulone, isoadhumulone,dihydro-isohumulone, dihydro-isocohumulone, dihydro-adhumulone,tetrahydro-isohumulone, tetrahydro-isocohumulone, tetrahydro-adhumulone,hexahydro-isohumulone, hexahydro-isocohumulone, andhexahydro-adhumulone. The preferred compounds can bear substituents, asshown in the formula above.

Another embodiment comprises composition containing tryptanthrin andconjugates thereof.

Other embodiments relate to combinations of components. Preferredcompositions can function to specifically inhibit COX-2 expression, tomodulate NFκB, to inhibit prostaglandin synthesis selectively in targetcells, or to inhibit inflammation response selectively in target cells,including synergistic effects.

One embodiment relates to compositions that include, as a firstcomponent, an active ingredient isolated or derived from an extract ofhops and as a second component at least one member selected from thegroup consisting of rosemary, an extract or compound derived fromrosemary, a triterpene species or derivatives or conjugates thereof, aditerpene lactone species or derivatives or conjugates thereof, andtryptanthrin or conjugates thereof. Another embodiment relates tocompositions that include, as a first component, tryptanthrin orconjugates thereof and as a second component at least one memberselected from the group consisting of an active ingredient isolated orderived from an extract of hops, rosemary, an extract or compoundderived from rosemary, a triterpene species or derivatives or conjugatesthereof, a diterpene lactone species or derivatives or conjugatesthereof.

Dosage

The selected dosage level will depend upon activity of the particularcomposition, the route of administration, the severity of the conditionbeing treated or prevented, and the condition and prior medical historyof the patient being treated. However, it is within the skill of the artto start doses of the composition at levels lower than required toachieve the desired therapeutic effect and to gradually increase thedosage until the desired effect is achieved. If desired, the effectivedaily dose may be divided into multiple doses for purposes ofadministration, e.g., two to four separate doses per day. It will beunderstood, however, that the specific dose level for any particularpatient will depend upon a variety of factors including body weight,general health, diet, time and route of administration, combination withother compositions and the severity of the particular condition beingtreated or prevented.

Preferred embodiments include delivering an effective amount of hopsfractions, hops compounds, or hops derivatives alone or with incombination with other active ingredients. Preferably, a daily dose ofpreferred compositions would be formulated to deliver about 0.5 to10,000 mg of alpha acid, isoalpha acid, reduced isoalpha acid,tetra-hydroisoalpha acid, hexa-hydroisoalpha acid, beta acid, or spenthops per day. More preferably, an effective daily dose of preferredcompositions would be formulated to deliver about 50 to 7500 mg, about100 to 5000 mg, about 200 to 3000 mg, or about 500 to 2000 mg of alphaacids, isoalpha acid, reduced isoalpha acid, tetra-hydroisoalpha acid,hexa-hydroisoalpha acid, beta acid, or spent hops per day. Preferably,the effective daily dose is administered once or twice a day. A certainembodiment provides a composition comprising about 0.5 to 800 mg ofisoalpha acid or reduced isoalpha acid, more preferably about 50 to 400mg or about 100 to 200 mg of isoalpha acid or reduced isoalpha acid perday. Another certain embodiment provides a composition comprising about10 to 3000 mg of reduced isoalpha acid, tetra-hydroisoalpha acid, orhexa-hydroisoalpha acid per day, more preferably about 50 to 2000 mg,about 100 to 1500 mg, or about 200 to 1000 mg, of reduced isoalpha acid,tetra-hydroisoalpha acid, or hexa-hydroisoalpha acid per day. Yetanother certain embodiment provides a composition comprising about 50 to7500 mg of spent hops per day, preferably about 100 to 6000 mg, about200 to 5000 mg, or about 500 to 3000 mg of spent hops per day.

Preferred embodiments include delivering an effective amount oftryptanthrin or conjugates thereof alone or with in combination withother active ingredients. Preferably, a daily dose of preferredcompositions would be formulated to deliver about 0.0005 to 50 mgtryptanthrin/kg body weight per day. More preferably, an effective dailydose of preferred compositions would be formulated to deliver about 0.01to 10 mg or about 0.1 to 5 mg tryptanthrin/kg body weight per day.Preferably, a daily dose of preferred compositions would be formulatedto deliver about 0.035 to 3500 mg of tryptanthrin per day. Morepreferably, an effective daily dose of preferred composition would beformulated to deliver about 0.7 to 700 mg, about 1 to 500 mg or about 10to 100 mg of tryptanthrin per day. Preferably, the effective daily doseis administered once or twice a day.

Preferred embodiments include delivering an effective amount of rosemaryor an extract or compound derived from rosemary in combination withother active ingredients. Preferably, a daily dose of preferredcompositions would be formulated to deliver about 0.5 to 5000 mg ofrosemary, an extract of rosemary, or rosemary-derived compound per day.More preferably, an effective daily dose of preferred composition wouldbe formulated to deliver about 5 to 2000 mg or about 100 to 1000 mg ofrosemary, an extract of rosemary, or rosemary-derived compound per day.Preferably, the effective daily dose is administered once or twice aday. A certain embodiment provides a composition comprising about 75 mgof rosemary extract or rosemary-derived compound or derivative, to beadministered once or twice a day.

Preferred embodiments include delivering an effective amount of atriterpene or diterpene lactone species or derivatives or conjugatesthereof in combination with other active ingredients. Preferably, adaily dose of preferred compositions would be formulated to deliverabout 0.0005 to 50 mg triterpene or diterpene lactone/kg body weight perday. More preferably, an effective daily dose of preferred compositionswould be formulated to deliver about 0.01 to 10 mg or about 0.1 to 1 mgtriterpene or diterpene lactone/kg body weight per day. Preferably, adaily dose of preferred compositions would be formulated to deliverabout 0.035 to 3500 mg of triterpene or diterpene lactone species perday. More preferably, an effective daily dose of preferred compositionwould be formulated to deliver about 0.7 to 700 mg of triterpene orditerpene lactone species per day. Preferably, the effective daily doseis administered once or twice a day.

Preferably, an embodiment provides a composition containing an extractof rosemary and a triterpene, such as oleanolic acid, along with anactive ingredient, such as a fraction isolated or derived from hops ortryptanthrin or conjugate thereof. Preferably, an embodiment provides acomposition comprising about 0.01 to 500 mg of rosemary extract andabout 0.01 to 500 mg of oleanolic acid. Preferably, an embodimentprovides a composition capable of producing concentrations in targettissues of 0.1 to 10 μg/g tissue of rosemary extract and about 0.1 to 25μg/g tissue of oleanolic acid.

A composition of preferred embodiments for topical application wouldcontain about 0.001 to 10 weight percent, preferably about 0.1 to 1weight percent of a hops extract component or derivative or tryptanthrinor conjugate thereof. Preferred embodiments would produce serumconcentrations in the ranges of about 0.0001 to 10 μM, preferably about0.01 to 1 μM of a fraction isolated or derived from hops or tryptanthrinor conjugate thereof. The preferred embodiments for topical applicationcan further comprise an additional ingredient selected from rosemary, anextract or compound derived from rosemary, a triterpene species orderivatives or conjugates thereof a diterpene lactone species orderivatives or conjugates thereof, a fraction isolated or derived fromhops or tryptanthrin or conjugates thereof at concentrations of eachcomponent of 0.001 to 10 weight percent, preferably 0.1 to 1 weightpercent Preferred embodiments would produce serum concentrations in theranges of about 0.001 to 50 μM, preferably about 0.1 μM to 5 μM of theadditional ingredient.

A certain composition comprises a first component selected from afraction isolated or derived from hops and a second component comprisingan extract or compound derived from rosemary, an extract or compoundderived from rosemary, a triterpene species or derivatives or conjugatesthereof a diterpene lactone species or derivatives or conjugatesthereof, or tryptanthrin or conjugates thereof. Preferably, the weightratio of the first component, i.e. a fraction isolated or derived fromhops to the second component, i.e. an extract or compound derived fromrosemary, an extract or compound derived from rosemary, a triterpenespecies or derivatives or conjugates thereof, a diterpene lactonespecies or derivatives or conjugates thereof or tryptanthrin orconjugates thereof, is within a range of about 100:1 to about 1:100;preferably about 50:1 to about 1:50; more preferably about 10:1 to about1:10.

A certain composition comprises a first component of tryptanthrin andconjugates thereof, and a second component comprising hops fraction,hops compound, hops derivative, rosemary, an extract or compound derivedfrom rosemary, a triterpene species or derivatives or conjugatesthereof, or a diterpene lactone species or derivatives or conjugatesthereof. Preferably, the weight ratio of the first component, i.e.tryptanthrin and conjugates thereof, to the second component, i.e. hopsfraction, hops compound, hops derivative, rosemary, an extract orcompound derived from rosemary, a triterpene species or derivatives orconjugates thereof, or a diterpene lactone species or derivatives orconjugates thereof, is within a range of about 100:1 to about 1:100;preferably about 50:1 to about 1:50; more preferably about 10:1 to about1:10; even more preferably about 1:1. It is understood that one skilledin art can readily use the amounts described above or appropriateintermediate amounts that are effective for a desired activity.

Applications of Preferred Compositions

As stated previously, the generally held concept (COX dogma) is thatCOX-1 is expressed constitutively in most tissues whereas COX-2 is theinducible enzyme triggered by pro-inflammatory stimuli includingmitogens, cytokines and bacterial lipopolysaccharide (LPS) in cells invitro and in inflamed sites in vivo. Based primarily on such differencesin expression, COX-1 has been characterized as a housekeeping enzyme andis thought to be involved in maintaining physiological functions such ascytoprotection of the gastric mucosa, regulation of renal blood flow,and control of platelet aggregation. COX-2 is considered to mainlymediate inflammation, although constitutive expression is found inbrain, kidney and the gastrointestinal tract. Therefore, it would bedesirable to down-regulate expression of COX-2 tissue-specifically orcell-specifically. Such down-regulation can be achieved by modulatingNFκB. Examples of target cells include, but are not limited to,inflammatory cells, pulmonary cells, microglia and tumor cells. Examplesof nontarget cells include, but are not limited to, gastric mucosal,neural, and renal cells.

The compositions have widespread applications. Preferred compositionscan be useful for treating conditions, such as cancer, autoimmunediseases, inflammatory diseases, neurological diseases. Preferredcompositions are also believed to be useful for treating conditions,such as HIV-1 infections, rhinovirus infections, and cardiovasculardiseases.

Preferred embodiments would be useful for, but not limited to a numberof inflammatory conditions and can include conditions associated withtissue-specific activation of NFκB. Thus, the invention includestreatment of inflammation in a subject, and treatment of otherinflammation-associated disorders, such as, as an analgesic in thetreatment of pain and headaches, or as an antipyretic for the treatmentof fever. Additional examples of such preferred embodiments would beuseful to treat arthritis, including but not limited to rheumatoidarthritis, spondyloathopathies, gouty arthritis, osteoarthritis,systemic lupus erythematosis, and juvenile arthritis. Such preferredembodiments would be useful in the treatment of asthma, bronchitis,menstrual cramps, tendonitis, bursitis, and skin related conditions suchas psoriasis, eczema, burns and dermatitis. Preferred embodiments alsowould be useful to treat gastrointestinal conditions such asinflammatory bowel disease, Crohn's disease, gastritis, irritable bowelsyndrome and ulcerative colitis and for the prevention or treatment ofcancer such as colorectal cancer. Preferred embodiments would be usefulin treating inflammation in such diseases as vascular diseases, migraineheadaches, periarteritis nodosa, thyroiditis, aplastic anemia, Hodgkin'sdisease, sclerodma, rheumatic fever, type I diabetes, myasthenia gravis,multiple sclerosis, sacoidosis, nephrotic syndrome, Behchet's syndrome,polymyositis, gingivitis, hypersensitivity, swelling occurring afterinjury, myocardial ischemia, periodontal disease, insulitis and thelike.

Preferred embodiments would also be useful in the treatment ofophthalmic diseases, such as retinopathies, conjunctivitis, uveitis,ocular photophobia, and of acute injury to the eye tissue. Preferredembodiments would also be useful in the treatment of pulmonaryinflammation, such as that associated with viral infections and cysticfibrosis. Preferred embodiments would also be useful in the treatment ofasthma. Preferred embodiments would also be useful for the treatment ofcertain nervous system disorders such as cortical dementias includingAlzheimer's disease. Preferred embodiments are useful asanti-inflammatory agents, such as for the treatment of arthritis, withthe additional benefit of having significantly less harmful sideeffects. As inhibitors of COX-2 mediated biosynthesis of PGE₂, thesecompositions would also be useful in the treatment of allergic rhinitis,respiratory distress syndrome, endotoxin shock syndrome,atherosclerosis, and central nervous system damage resulting fromstroke, ischemia and trauma. The preferred embodiments would also beuseful for the treatment of fibromyalgia.

Since COX-2 can also play a role in the regulation of osteoblasticfunction, preferred embodiments can also be useful for treating andpreventing osteoporosis. Kanematsu et al. (J Bone Miner Res 1997November; 12(11):1789-96.) discloses that interleukin 1 (IL-1) and tumornecrosis factor alpha (TNF-alpha) have been implicated in thepathogenesis of osteoporosis. These proinflammatory cytokines induceboth COX-2 and nitric oxide synthase (iNOS) with the release of PGE₂ andNO, respectively. They determined the interaction between COX and NOSpathways and their role in the regulation of osteoblastic function inMC3T3-E1 cells.

According to preferred embodiments, the animal may be a member selectedfrom the group consisting of humans, non-human primates, dogs, cats,birds, horses, ruminants or other warm blooded animals. Preferredembodiments are directed primarily to the treatment of human beings.Administration can be by any method available to the skilled artisan,for example, by oral, topical, transdermal, transmucosal, or parenteralroutes.

Besides being useful for human treatment, preferred embodiments are alsouseful for treatment of other animals, including horses, dogs, cats,birds, sheep, pigs, etc. A certain formulation for the treatment ofinflammation would inhibit the induction and activity of COX-2 withlittle effect on the synthesis of PGE₂ in the gastric mucosa.Historically, the NSAIDs used for treatment of inflammation lacked thespecificity of inhibiting COX-2 without affecting PGE₂ synthesis ingastric mucosal cells. Therefore, these drugs irritated and damaged thegastrointestinal system when used for extended periods.

Preferred compositions can also modulate NF-κB. Modulation of NF-κB caninclude regulating levels of NF-κB to treat or inhibit a pathologicalcondition in a mammal. For example, abnormal levels, such as increasedlevels, of NF-κB can be associated with diseases and undesirableconditions. NF-κB is implicated in neuronal survival, inflammatoryresponse, and cancer. NF-κB regulates COX-2 gene expression. Therefore,preferred compositions that modulate NF-κB can also affect theexpression of COX-2.

Results presented herein indicate that modulation of NF-κB results inmodulation of COX-2 expression in target cells only, without anysignificant direct inhibition of COX-2 or other enzymes with PG pathway.Therefore, for example, preferred compositions offer the advantage ofanti-inflammatory effects without the side effects of damaging gastricmucosa, which are present in many existing NSAIDs. Existing NSAIDs, suchas rofecoxib and celxobib, are supposed to inhibit the synthesis ofprostanglandins by selectively inhibiting the COX-2 enzyme. However,side effects still occur with these existing NSAIDs because of lack oftotal selective inhibition of COX-2. Existing NSAIDs can still bepromiscuous and affect enzymes other than COX-2 to result in sideeffects. By modulating NF-κB, compositions of preferred embodiments actat an upstream position and can inhibit the synthesis of COX-2selectively in target cells. Without COX-2 in target cells, thesynthesis of prostaglandins directed to inflammatory reactions can alsobe inhibited. Therefore, the inflammatory reaction can be prevented orhalted. While COX-2 in target cells is affected, COX-1 and COX-2 innontarget cells remain unaffected and continues to maintainphysiological functions, such as cytoprotection of gastric mucosa,regulation of renal blood flow, and control of platelet aggregation.

Since preferred compositions can affect NF-κB, preferred embodiments canalso be useful for treating and preventing a variety of disordersincluding, but not limited to, autoimmune, inflammatory, neurologicaland cardiovascular diseases, and cancer.

Preferred embodiments can be useful for treating and preventing apathological condition associated with tissue-specific activation ofNF-κB. NF-κB can be found in numerous cell types and is found to beactivated by a wide range of inducers. Upon activation and transport tothe nuclei, NF-κB can initiate or regulate early-response genetranscription by binding to motifs found in the promoter or enhancerregions of specific genes.

The NF-κB response occurs in virtually all cell types in combinationwith a variety of co-activators. However, because NF-κB alone is notcapable of activating its genes when bound to DNA, the exact genesactivated will vary depending on the cellular context. Co-activators arebelieved to link enhancer-bound transcription factors, like NF-κB, tocomponents of the basal transcription machinery, which then transcribethe gene to generate the mRNA copy. Therefore, NF-κB can be modulatedtissue- or cell-specifically so as to treat and/or inhibit a variety ofpathological conditions.

Therefore, compositions that inhibit or activate specific targets in theNF-κB pathway provide new approaches in the treatment or prevention of anumber of serious diseases, including cancer and inflammatory disorders.NF-κB is a transcription factor that is involved in a range of cellularphenomena, including inflammation, antigen presentation, immunity,cytokine production, apoptosis and cancer. For example, as NF-κB affectspulmonary cells, a pathological condition can be manifested as asthma,and/or other pulmonary conditions. NF-κB is also involved in colorectal,mammary, and prostate conditions, such as cancer, as mediated by PGE₂.NF-κB is involved in these cancers and other cancers, as mediated bycell-to-cell adhesion. NF-κB is also involved in HIV-1 replication,colds, and flus. As stated above, NF-κB has been implicated inconditions, such as autoimmune, inflammatory, neurological andcardiovascular diseases, and cancer.

Formulations

Preferred compositions can be administered in the form of a dietarysupplement or therapeutic composition. The compositions may beadministered orally, topically, transdermally, transmucosally,parenterally, etc., in appropriate dosage units, as desired.

Preferred compositions for dietary application may include variousadditives such as other natural components of intermediary metabolism,vitamins and minerals, as well as inert ingredients such as talc andmagnesium stearate that are standard excipients in the manufacture oftablets and capsules. For example, one embodiment comprises activeingredients of preferred compositions in combination with glucosamine orchondrotin sulfate.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, isotonic and absorptiondelaying agents, sweeteners and the like. These pharmaceuticallyacceptable carriers may be prepared from a wide range of materialsincluding, but not limited to, diluents, binders and adhesives,lubricants, disintegrants, coloring agents, bulking agents, flavoringagents, sweetening agents and miscellaneous materials such as buffersand absorbents that may be needed in order to prepare a particulartherapeutic composition. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredients, its use in preferred compositions is contemplated.In one embodiment, talc, and magnesium stearate are included in theformulation. Other ingredients known to affect the manufacture of thiscomposition as a dietary bar or functional food can include flavorings,sugars, amino-sugars, proteins and/or modified starches, as well as fatsand oils.

Dietary supplements, lotions or therapeutic compositions of preferredembodiments can be formulated in any manner known by one of skill in theart. In one embodiment, the composition is formulated into a capsule ortablet using techniques available to one of skill in the art. In capsuleor tablet form, the recommended daily dose for an adult human or animalwould preferably be contained in one to six capsules or tablets.However, preferred compositions can also be formulated in otherconvenient forms, such as an injectable solution or suspension, a spraysolution or suspension, a lotion, gum, lozenge, food or snack item.Food, snack, gum or lozenge items can include any ingestible ingredient,including sweeteners, flavorings, oils, starches, proteins, fruits orfruit extracts, vegetables or vegetable extracts, grains, animal fats orproteins. Thus, preferred compositions can be formulated into cereals,snack items such as chips, bars, gumdrops, chewable candies or slowlydissolving lozenges. Preferred embodiments contemplate treatment of alltypes of inflammation-based diseases, both acute and chronic. Preferredformulations reduce the inflammatory response and thereby promoteshealing of, or prevents further damage to, the affected tissue. Apharmaceutically acceptable carrier can also be used in the preferredcompositions and formulations.

Assay Using AGS Cell Line

In order to identify selective COX-2 drugs, it has been common practiceto use the Modified Whole Blood/Cell Assay of T. D. Warner et al.,Nonsteroid drug selectivities for cyclooxygenase-1 rather thancyclooxygenase-2 are associated with human gastrointestinal toxicity: Afull in vitro analysis, Proc. Natl. Sci. USA 96:7563-7568(1999). Whenhop fractions are tested according to this procedure, hops extracts donot yield IC₅₀ values in the necessary μg/mL range, since they are notdirect inhibitors of COX-2. This lack of direct inhibition of COX-2 wasdemonstrated by Tobe, H. et al. 1997. (Bone resorption Inhibitors fromhop extract. Biosci. Biotech. Biochem 61(1)158-159) using purified COX-2enzyme. Similarly, EXAMPLE 4 of this application demonstrates that, whentested according to the Modified Whole Blood/Cell Assay, hops compoundsand derivatives produce median inhibitory concentrations greater than 25μg/mL. Such high median inhibitory concentrations are pharmacologicallyunsuitable. Therefore, the Modified Whole Blood Assay as described byWarner is an invalid procedure for formulating potentiallytherapeutically effective combinations containing hops or hopsderivatives.

The discovery of COX-2 has made possible the design of drugs that reduceinflammation without removing the protective PGs in the stomach andkidney made by COX-1. One of our approaches is to screen compositions ofthe preferred embodiments using in vitro animal cells to assess COX-2and COX-1 inhibitory activity employing PGE₂, which has cytoprotectiveactions and play a role in maintaining the integrity of thegastrointestinal mucosa, as an endpoint. Secondarily, different celltypes are used to confirm results. The screening process would indicatecompositions that have specific COX-2 activity and limited COX-1inhibition. Compositions of preferred embodiments can be tested in twocell types: 1) human pulmonary cells or other cell line to determine andidentify optimal amounts and ratios for compositions comprising morethan one component; and 2) human gastric epithelial cells (AGS cellline), a gastrointestinal tract cell line and a model system forassessing toxicity which is typically related to inhibition of COX-1which is required for wound healing (such as ulcers). Hence,compositions of preferred embodiments that can inhibit COX-2 or COX-2induction can be screened by selecting compositions that have low or noactivity in AGS cells and good activity in human pulmonary cells orother cell line.

In particular embodiments, the invention provides a compositioncomprising, as a first component, a fraction derived from hops; and as asecond component, at least one member selected from the group consistingof rosemary, an extract derived from rosemary, a compound derived fromrosemary, a triterpene species, a diterpene lactone species, andtryptanthrin. The fraction derived from hops can be extracted with CO₂.The fraction derived from hops can also be selected from the groupconsisting of isoalpha acids, reduced isoalpha acids,tetra-hydroisoalpha acids, hexa-hydroisoalpha acids, and spent hops.

In another embodiment, a composition of the invention can contain afraction derived from hops comprising a compound of a supragenus havingthe formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is allyl;    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; and    -   wherein R, T, X, and Z are independently selected from the group        consisting of H, F, Cl, Br, I, and π orbital, with the proviso        that if one of R, T, X, or Z is a π orbital, then the adjacent        R, T, X, or Z is also a π orbital, thereby forming a double        bond.

In yet another embodiment, the fraction derived from hops can comprise acompound of Genus A having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In yet another embodiment, the fraction derived from hops can comprise acompound of Genus B having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In a composition of the invention, the fraction derived from hops cancomprise a compound selected from the group consisting of cohumulone,adhumulone, isohumulone, isocohumulone, isoadhumulone,dihydro-isohumulone, dihydro-isocohumulone, dihydro-adhumulone,tetrahydro-isohumulone, tetrahydro-isocohumulone, tetrahydro-adhumulone,hexahydro-isohumulone, hexahydro-isocohumulone, andhexahydro-adhumulone.

In a composition of the invention, a second component can be a compoundderived from rosemary that is selected from the group consisting of1,8-cineole, 19-alpha-hydroxyursolic acid, 2-β-hydroxyoleanolic acid,3-O-acetyloleanolic acid, 3-O-acetylursolic acid,6-methoxy-luteolin-7-glucoside, 6-methoxyluteolin,6-methoxyluteolin-7-glucoside, methoxyluteolin-7-methylether,7-ethoxy-rosmanol, 7-methoxy-rosmanol, alpha-amyrin, alpha-humulene,alpha-hydroxyhydrocaffeic acid, alpha-pinene, alpha-terpinene,alpha-terpinenyl acetate, alpha-terpineol, alpha-thujone, apigenin,apigenin-7-glucoside, curcumene, benzyl-alcohol, β-amyrenone, β-amyrin,β-elemene, β-pinene, betulin, betulinic acid, borneol, bornyl-acetate,caffeic acid, camphene, camphor, carnosic acid, carnosol, carvacrol,carvone, caryophyllene, caryophyllene-oxide, chlorogenic acid,diosmetin, gamma-terpinene, hesperidin, isoborneol, limonene, luteolin,luteolin-3′-O-(3″-O-acetyl)-β-D-glucuronide,luteolin-3′-O-(4″-O-acetyl)-β-D-glucuronide,luteolin-3′-O-β-D-glucuronide, luteolin-7-glucoside, methyl-eugenol,myrcene, neo-chlorogenic acid, nepetin, octanoic acid, oleanolic acid,p-cymene, piperitenone, rosmanol, rosmaric acid, rosmaricine,rosmaridiphenol, rosemarinic acid, rosmarinol, rosmariquinone, sabinene,sabinyl acetate, salicylates, salicylic acid-2-β-D-glucoside, squalene,terpinen-4-ol, terpinolene, thymol, trans-anethole, trans-carveol,ursolic acid, verbenone, and zingiberene.

In another embodiment, the second component can be a compound derivedfrom rosemary that is selected from the group consisting of betulin,betulinic acid, carnosic acid, carnosol, carvacrol, chlorogenic acid,diosmetin, limonene, and luteolin. In still a further embodiment, thesecond component can be a triterpene species that is selected from thegroup consisting of 18-a-glycyrrhetinic acid, 18-β-glycyrrhetinic acid,2-a-3-a-dihydrooxyurs-12-3n-28-onic acid, 3-a-hydroxyursolic acid,3-oxo-ursolic acid, betulin, betulinic acid, celastrol, eburicoic acid,friedelin, glycyrrhizin, gypsogenin, oleanolic acid, oleanolicacid-3-acetate, pachymic acid, pinicolic acid, sophoradiol,soyasapogenol A, soyasapogenol B, tripterin, triptophenolide, tumulosicacid, ursolic acid, ursolic acid-3-acetate, uvaol, and β-sitosterol.Furthermore, the second component can be a triterpene species that isselected from the group consisting of 18-a-glycyrrhetinic acid,18-β-glycyrrhetinic acid, 2-a-3-a-dihydrooxyurs-12-3n-28-onic acid,3-a-hydroxyursolic acid, 3-oxo-ursolic acid, betulin, betulinic acid,celastrol, friedelin, oleanolic acid, tripterin, triptophenolide,ursolic acid, and uvaol. In addition, the second component can betryptanthrin, a triterpene species, or a diterpene lactone species thatis conjugated to a member selected from the group consisting of mono- ordi-saccharides, amino acids, sulfates, succinate, acetate, andglutathione.

A composition of the invention can also comprise about 0.5 to 10000 mgof the fraction isolated or derived from hops or about 50 to 7500 mg ofthe fraction isolated or derived from hops. A composition of theinvention can additionally comprise about 0.035 to 3500 mg oftryptanthrin, or about 0.7 to 700 mg of tryptanthrin, wherein the secondcomponent is tryptanthrin. A composition of the invention can alsocomprise about 0.5 to 5000 mg of the second component, or about 5 to2000 mg of the second component, wherein the second component isselected from the group consisting of rosemary, extract derived fromrosemary, and a compound derived from rosemary. Additionally, acomposition of the invention can comprise about 0.035 to 3500 mg of atriterpene species, or about 0.7 to 700 mg of a triterpene species,wherein the second component is a triterpene species.

In still another embodiment, a composition can comprise about 0.001 to10 weight percent of the first component, or about 0.1 to 1 weightpercent of the first component. In addition, a composition can compriseabout 0.001 to 10 weight percent of the second component, or about 0.1to 1 weight percent of the second component. In a composition of theinvention, the ratio of the first component to the second component canbe in the range of about 100:1 to about 1:100, or about 50:1 to about1:50. Any of the compositions of the invention can further comprise apharmaceutically acceptable carrier, and such a composition comprising apharmaceutically acceptable carrier can be used in the methods of theinvention.

The invention also provides a composition comprising as a firstcomponent, a fraction isolated or derived from hops; and as a secondcomponent, at least one member selected from the group consisting ofrosemary, an extract derived from rosemary, a compound derived fromrosemary, and tryptanthrin. The fraction isolated or derived from hopscan be extracted with CO₂. The fraction isolated or derived from hopscan be selected from the group consisting of alpha acids, isoalphaacids, reduced isoalpha acids, tetra-hydroisoalpha acids,hexa-hydroisoalpha acids, beta acids, and spent hops.

The fraction isolated or derived from hops can comprise a compound of asupragenus having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl;    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; and    -   wherein R, T, X, and Z are independently selected from the group        consisting of H, F, Cl, Br, I, and π orbital, with the proviso        that if one of R, T, X or Z is a π orbital, then the adjacent R,        T, X, or Z is also a π orbital, thereby forming a double bond.

The fraction isolated or derived from hops can also comprise a compoundof Genus A having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

The fraction isolated or derived from hops can additionally comprise acompound of Genus B having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

The fraction isolated or derived from hops can additionally comprise acompound selected from the group consisting of humulone, cohumulone,adhumulone, isohumulone, isocohumulone, isoadhumulone,dihydro-isohumulone, dihydro-isocohumulone, dihydro-adhumulone,tetrahydro-isohumulone, tetrahydro-isocohumulone, tetrahydro-adhumulone,hexahydro-isohumulone, hexahydro-isocohumulone, andhexahydro-adhumulone.

Furthermore, the second component of such a composition can be acompound derived from rosemary that is selected from the groupconsisting of 1,8-cineole, 19-alpha-hydroxyursolic acid,2-β-hydroxyoleanolic acid, 3-O-acetyloleanolic acid, 3-O-acetylursolicacid, 6-methoxy-luteolin-7-glucoside, 6-methoxyluteolin,6-methoxyluteolin-7-glucoside, methoxyluteolin-7-methylether,7-ethoxy-rosmanol, 7-methoxy-rosmanol, alpha-amyrin, alpha-humulene,alpha-hydroxyhydrocaffeic acid, alpha-pinene, alpha-terpinene,alpha-terpinenyl acetate, alpha-terpineol, alpha-thujone, apigenin,apigenin-7-glucoside, curcumene, benzyl-alcohol, β-amyrenone, β-amyrin,β-elemene, β-pinene, betulin, betulinic acid, borneol, bornyl-acetate,caffeic acid, camphene, camphor, carnosic acid, carnosol, carvacrol,carvone, caryophyllene, caryophyllene-oxide, chlorogenic acid,diosmetin, gamma-terpinene, hesperidin, isoborneol, limonene, luteolin,luteolin-3′-O-(3″-O-acetyl)-β-D-glucuronide,luteolin-3′-O-(4″-O-acetyl)-β-D-glucuronide,luteolin-3′-β-D-glucuronide, luteolin-7-glucoside, methyl-eugenol,myrcene, neo-chlorogenic acid, nepetin, octanoic acid, oleanolic acid,p-cymene, piperitenone, rosmanol, rosmaric acid, rosmaricine,rosmaridiphenol, rosemarinic acid, rosmarinol, rosmariquinone, sabinene,sabinyl acetate, salicylates, salicylic acid-2-β-D-glucoside, squalene,terpinen-4-ol, terpinolene, thymol, trans-anethole, trans-carveol,ursolic acid, verbenone, and zingiberene. The second component can alsobe a compound derived from rosemary that is selected from the groupconsisting of betulin, betulinic acid, carnosic acid, carnosol,carvacrol, chlorogenic acid, diosmetin, limonene, and luteolin.Additionally, the second component can be tryptanthrin that isconjugated to a member selected from the group consisting of mono- ordi-saccharides, amino acids, sulfates, succinate, acetate, andglutathione.

In a particular embodiment, the composition can comprise about 0.5 to10000 mg or about 50 to 7500 mg of the fraction isolated or derived fromhops. In addition, the composition can comprise about 0.35 to 3500 mg oftryptanthrin, or about 0.7 to 700 mg of tryptanthrin, wherein the secondcomponent is tryptanthrin. Moreover, the composition can comprise about0.5 to 5000 mg of the second component, or about 5 to 2000 mg of thesecond component, wherein the second component is selected from thegroup consisting of rosemary, extract derived from rosemary, and acompound derived from rosemary. In addition, the composition cancomprise about 0.001 to 10 weight percent of the first component, orabout 0.1 to 1 weight percent of the first component. Also, thecomposition can comprise about 0.001 to 10 weight percent of the secondcomponent, about 0.1 to 1 weight percent of the second component. Inanother embodiment, a ratio of the first component to the secondcomponent can be in the range of about 100:1 to about 1:100, or in therange of about 50:1 to about 1:50. The composition can further comprisea pharmaceutically acceptable carrier.

In still another embodiment, the invention provides a method ofmodulating inflammatory response in cells, the method comprisingcontacting the cells with a composition of the invention. For example,the method can be carried out using a composition comprising a fractionisolated or derived from hops and a second component selected from thegroup consisting of rosemary, an extract derived from rosemary, acompound derived from rosemary, a triterpene species, a diterpenelactone species, and tryptanthrin. The invention also provides a methodof treating or inhibiting a pathological condition in a mammalassociated with tissue-specific activation of inflammation, the methodcomprising administering to the mammal a composition comprising afraction isolated or derived from hops and a second component selectedfrom the group consisting of rosemary, an extract derived from rosemary,a compound derived from rosemary, a triterpene species, a diterpenelactone species, and tryptanthrin.

In such a method of treating or inhibiting a pathological condition, thecomposition can contain a fraction isolated or derived from hops can beselected from the group consisting of alpha acids, isoalpha acids,reduced isoalpha acids, tetra-hydroisoalpha acids, hexa-hydroisoalphaacids, beta acids, and spent hops. In another embodiment of the method,the fraction isolated or derived from hops comprises a compound of asupragenus having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl;    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; and    -   wherein R, T, X, and Z are independently selected from the group        consisting of H, F, Cl, Br, L and π orbital, with the proviso        that if one of R, T, X, or Z is a π orbital, then the adjacent        R, T, X, or Z is also a π orbital, thereby forming a double        bond.

In the method, the fraction isolated or derived from hops can alsocomprise a compound of Genus A having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In yet another embodiment of the method, the fraction isolated orderived from hops can additionally comprise a compound of Genus B havingthe formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In still another embodiment of the method, the fraction isolated orderived from hops comprises a compound selected from the groupconsisting of humulone, cohumulone, adhumulone, isohumulone,isocohumulone, isoadhumulone, dihydro-isohumulone,dihydro-isocohumulone, dihydro-adhumulone, tetrahydro-isohumulone,tetrahydro-isocohumulone, tetrahydro-adhumulone, hexahydro-isohumulone,hexahydro-isocohumulone, and hexahydro-adhumulone. In a particularembodiment, the composition can comprise about 0.5 to 10000 mg or about50 to 7500 mg of the fraction isolated or derived from hops.Furthermore, the composition can comprise about 0.001 to 10 weightpercent or about 0.1 to 1 weight percent of the fraction isolated orderived from hops. In a particular embodiment, the second component isrosemary. In another embodiment the second component is an extractderived from rosemary. In still another embodiment, the second componentis a triterpene species. In a method of the invention, the compositioncan further comprise a third component different from the secondcomponent, where the third component is selected from the groupconsisting of rosemary, an extract derived from rosemary, a compoundderived from rosemary, a triterpene species, a diterpene lactonespecies, and tryptanthrin. In a particular embodiment, the second andthird components are an extract derived from rosemary and tryptanthrin,respectively.

In another embodiment of the method, the second component can be acompound derived from rosemary that is selected from the groupconsisting of 1,8-cineole, 19-alpha-hydroxyursolic acid,2-β-hydroxyoleanolic acid, 3-O-acetyloleanolic acid, 3-O-acetylursolicacid, 6-methoxy-luteolin-7-glucoside, 6-methoxyluteolin,6-methoxyluteolin-7-glucoside, methoxyluteolin-7-methylether,7-ethoxy-rosmanol, 7-methoxy-rosmanol, alpha-amyrin, alpha-humulene,alpha-hydroxyhydrocaffeic acid, alpha-pinene, alpha-terpinene,alpha-terpinenyl acetate, alpha-terpineol, alpha-thujone, apigenin,apigenin-7-glucoside, curcumene, benzyl-alcohol, β-amyrenone, β-amyrin,β-elemene, β-pinene, betulin, betulinic acid, borneol, bornyl-acetate,caffeic acid, camphene, camphor, carnosic acid, carnosol, carvacrol,carvone, caryophyllene, caryophyllene-oxide, chlorogenic acid,diosmetin, gamma-terpinene, hesperidin, isoborneol, limonene, luteolin,luteolin-3′-O-(3″-O-acetyl)-β-D-glucuronide,luteolin-3′-O-(4″-O-acetyl)-β-D-glucuronide,luteolin-3′-O-β-D-glucuronide, luteolin-7-glucoside, methyl-eugenol,myrcene, neo-chlorogenic acid, nepetin, octanoic acid, oleanolic acid,p-cymene, piperitenone, rosmanol, rosmaric acid, rosmaricine,rosmaridiphenol, rosemarinic acid, rosmarinol, rosmariquinone, sabinene,sabinyl acetate, salicylates, salicylic acid-2-β-D-glucoside, squalene,terpinen-4-ol, terpinolene, thymol, trans-anethole, trans-carveol,ursolic acid, verbenone, and zingiberene.

In such a method of the invention, the second component can also be acompound derived from rosemary that is selected from the groupconsisting of betulin, betulinic acid, carnosic acid, carnosol,carvacrol, chlorogenic acid, diosmetin, limonene, and luteolin. Thecomposition used in the method can comprise about 0.5 to 5000 mg of thesecond component, or about 5 to 2000 mg of the second component, whereinthe second component is selected from the group consisting of rosemary,extract derived from rosemary, and a compound derived from rosemary. Instill another embodiment, the second component used in a method of theinvention can be a triterpene species or a diterpene lactone speciesthat is conjugated to a member selected from the group consisting ofmono- or di-saccharides, amino acids, sulfates, succinate, acetate, andglutathione.

In yet another embodiment of a method of the invention, the secondcomponent can be a triterpene species that is selected from the groupconsisting of 18-a-glycyrrhetinic acid, 18-β-glycyrrhetinic acid,2-a-3-a-dihydrooxyurs-12-3n-28-onic acid, 3-a-hydroxyursolic acid,3-oxo-ursolic acid, betulin, betulinic acid, celastrol, eburicoic acid,friedelin, glycyrrhizin, gypsogenin, oleanolic acid, oleanolicacid-3-acetate, pachymic acid, pinicolic acid, sophoradiol,soyasapogenol A, soyasapogenol B, tripterin, triptophenolide, tumulosicacid, ursolic acid, ursolic acid-3-acetate, uvaol, and β-sitosterol. Inaddition, the second component can be a triterpene species that isselected from the group consisting of 18-a-glycyrrhetinic acid,18-β-glycyrrhetinic acid, 2-a-3-a-dihydrooxyurs-12-3n-28-onic acid,3-a-hydroxyursolic acid, 3-oxo-ursolic acid, betulin, betulinic acid,celastrol, friedelin, oleanolic acid, tripterin, triptophenolide,ursolic acid, and uvaol.

In a particular embodiment of a method of the invention, the compositioncan comprise about 0.035 to 3500 mg of a triterpene species or about 0.7to 700 mg of a triterpene species, wherein the second component is atriterpene species. In another embodiment of the method, the secondcomponent is tryptanthrin that is conjugated to a member selected fromthe group consisting of mono- or di-saccharides, amino acids, sulfates,succinate, acetate, and glutathione. In still another embodiment of amethod of the invention, the composition can comprise about 0.035 to3500 mg of tryptanthrin, or about 0.7 to 700 mg of tryptanthrin, whereinthe second component is tryptanthrin. In addition, the composition usedin a method can comprise about 0.001 to 10 weight percent of the secondcomponent or about 0.1 to 1 weight percent of the second component.Furthermore, a ratio of the first component to the second component canbe in the range of about 100:1 to about 1:100 or in the range of about50:1 to about 1:50.

In such a method of treating or inhibiting a pathological condition, thepathological condition can be selected from the group consisting ofautoimmune diseases, inflammatory diseases, neurological diseases, andcancer. In addition, the pathological condition can be selected from thegroup consisting of inflammation, inflammation-associated disorders,arthritis, asthma, bronchitis, menstrual cramps, tendonitis, bursitis,skin-related conditions, gastrointestinal conditions, cancer, ophthalmicdiseases, pulmonary inflammation, nervous system disorders, allergicrhinitis, respiratory distress syndrome, endotoxin shock syndrome,atherosclerosis, and central nervous damage. In methods of theinvention, the composition can be administered in a variety of ways,including orally, topically, parenterally, or rectally.

The invention further provides a method of modulating the amount ofcyclooxygenase-2 (COX-2) activity in target cells without substantiallymodulating COX-2 activity in non-target cells, the method comprisingcontacting the cells with a composition comprising a fraction isolatedor derived from hops and a second component selected from the groupconsisting of rosemary, an extract derived from rosemary, a compoundderived from rosemary, a triterpene species, a diterpene lactonespecies, and tryptanthrin. In such a method of the invention, thenon-target cells can also be contacted with a fraction isolated orderived from hops. The contacting step can be performed in vivo. In amethod of the invention, the COX-2 activity can be modulated byinhibition of the COX-2 gene.

Additionally, the invention provides a method of treating or inhibitinga pathological condition in a mammal involving inhibiting inducibilityor activity of cyclooxygenase-2 (COX-2), the method comprisingadministering to the mammal a composition comprising a fraction isolatedor derived from hops and a second component selected from the groupconsisting of rosemary, an extract derived from rosemary, a compoundderived from rosemary, a triterpene species, a diterpene lactone, andtryptanthrin. In such a method of the invention, the fraction isolatedor derived from hops can be selected from the group consisting of alphaacids, isoalpha acids, reduced isoalpha acids, tetra-hydroisoalphaacids, hexa-hydroisoalpha acids, beta acids, and spent hops.

If desired in such a method of the invention, the fraction isolated orderived from hops comprises a compound of a supragenus having theformula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl;    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; and    -   wherein R, T, X, and Z are independently selected from the group        consisting of H, F, Cl, Br, I, and π orbital, with the proviso        that if one of R, T, X, or Z is a π orbital, then the adjacent        R, T, X, or Z is also a π orbital, thereby forming a double        bond.

In such a method of the invention, the fraction isolated or derived fromhops can also comprise a compound of Genus A having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In such a method of the invention, the fraction isolated or derived fromhops can also comprise a compound of Genus B having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In such a method of the invention, the fraction isolated or derived fromhops can comprise a compound selected from the group consisting ofhumulone, cohumulone, adhumulone, isohumulone, isocohumulone,isoadhumulone, dihydro-isohumulone, dihydro-isocohumulone,dihydro-adhumulone, tetrahydro-isohumulone, tetrahydro-isocohumulone,tetrahydro-adhumulone, hexahydro-isohumulone, hexahydro-isocohumulone,and hexahydro-adhumulone. In addition, the second component can be anextract derived from rosemary. Furthermore, the second component can bea triterpene species.

In another embodiment of a method of the invention, the compositionfurther can comprise a third component different from the secondcomponent, the third component being selected from the group consistingof rosemary, an extract derived from rosemary, a compound derived fromrosemary, a triterpene species, a diterpene lactone, and tryptanthrin.In a particular embodiment, the second and third components are anextract derived from rosemary and tryptanthrin, respectively.

In yet another embodiment of such a method of the invention, the secondcomponent is a compound derived from rosemary that is selected from thegroup consisting of 1,8-cineole, 19-alpha-hydroxyursolic acid,2-β-hydroxyoleanolic acid, 3-O-acetyloleanolic acid, 3-O-acetylursolicacid, 6-methoxy-luteolin-7-glucoside, 6-methoxyluteolin,6-methoxyluteolin-7-glucoside, methoxyluteolin-7-methylether,7-ethoxy-rosmanol, 7-methoxy-rosmanol, alpha-amyrin, alpha-humulene,alpha-hydroxyhydrocaffeic acid, alpha-pinene, alpha-terpinene,alpha-terpinenyl acetate, alpha-terpineol, alpha-thujone, apigenin,apigenin-7-glucoside, curcumene, benzyl-alcohol, β-amyrenone, β-amyrin,β-elemene, β-pinene, betulin, betulinic acid, borneol, bornyl-acetate,caffeic acid, camphene, camphor, carnosic acid, carnosol, carvacrol,carvone, caryophyllene, caryophyllene-oxide, chlorogenic acid,diosmetin, gamma-terpinene, hesperidin, isoborneol, limonene, luteolin,luteolin-3′-O-(3″-O-acetyl)-β-D-glucuronide,luteolin-3′-O-(4″-O-acetyl)-β-D-glucuronide,luteolin-3′-O-β-D-glucuronide, luteolin-7-glucoside, methyl-eugenol,myrcene, neo-chlorogenic acid, nepetin, octanoic acid, oleanolic acid,p-cymene, piperitenone, rosmanol, rosmaric acid, rosmaricine,rosmaridiphenol, rosemarinic acid, rosmarinol, rosmariquinone, sabinene,sabinyl acetate, salicylates, salicylic acid-2-β-D-glucoside, squalene,terpinen-4-ol, terpinolene, thymol, trans-anethole, trans-carveol,ursolic acid, verbenone, and zingiberene.

In another embodiment of such a method of the invention, the secondcomponent can be a triterpene species or a diterpene lactone speciesthat is conjugated to a member selected from the group consisting ofmono- or di-saccharides, amino acids, sulfates, succinate, acetate, andglutathione. Additionally, the second component can be a triterpenespecies that is selected from the group consisting of18-a-glycyrrhetinic acid, 18-β-glycyrrhetinic acid,2-a-3-a-dihydrooxyurs-12-3n-28-onic acid, 3-a-hydroxyursolic acid,3-oxo-ursolic acid, betulin, betulinic acid, celastrol, eburicoic acid,friedelin, glycyrrhizin, gypsogenin, oleanolic acid, oleanolicacid-3-acetate, pachymic acid, pinicolic acid, sophoradiol,soyasapogenol A, soyasapogenol B, tripterin, triptophenolide, tumulosicacid, ursolic acid, ursolic acid-3-acetate, uvaol, and β-sitosterol.Also, the second component can be tryptanthrin that is conjugated to amember selected from the group consisting of mono- or di-saccharides,amino acids, sulfates, succinate, acetate, and glutathione. If desiredin such a method of the invention, a ratio of the first component to thesecond component can be in the range of about 100:1 to about 1:100 or inthe range of about 50:1 to about 1:50.

In a particular embodiment of a method of the invention, thepathological condition involving inhibiting inducibility or activity ofCOX-2 can be selected from the group consisting of inflammation,inflammation-associated disorders, arthritis, asthma, bronchitis,menstrual cramps, tendonitis, bursitis, skin-related conditions,gastrointestinal conditions, cancer, ophthalmic diseases, pulmonaryinflammation, nervous system disorders, allergic rhinitis, respiratorydistress syndrome, endotoxin shock syndrome, atherosclerosis, andcentral nervous damage.

Also, the invention provides a method of inhibiting prostaglandinsynthesis selectively in target cells, the method comprising contactingthe cells with a fraction isolated or derived from hops and a secondcomponent selected from the group consisting of rosemary, an extractderived from rosemary, a compound derived from rosemary, a triterpenespecies, a diterpene lactone, and tryptanthrin. In such a method of theinvention, the fraction isolated or derived from hops can be selectedfrom the group consisting of alpha acids, isoalpha acids, reducedisoalpha acids, tetra-hydroisoalpha acids, hexa-hydroisoalpha acids,beta acids, and spent hops.

In addition in such a method of the invention, the fraction isolated orderived from hops can comprise a compound of a supragenus having theformula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl;    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; and    -   wherein R, T, X, and Z are independently selected from the group        consisting of H, F, Cl, Br, I, and π orbital, with the proviso        that if one of R, T, X, or Z is a π orbital, then the adjacent        R, T, X, or Z is also a π orbital, thereby forming a double        bond.

In another embodiment of the method of the invention, the fractionisolated or derived from hops can comprise a compound of Genus A havingthe formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In still another embodiment of such a method of the invention, thefraction isolated or derived from hops comprises a compound of Genus Bhaving the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In another embodiment of such a method of the invention, the fractionisolated or derived from hops can comprise a compound selected from thegroup consisting of humulone, cohumulone, adhumulone, isohumulone,isocohumulone, isoadhumulone, dihydro-isohumulone,dihydro-isocohumulone, dihydro-adhumulone, tetrahydro-isohumulone,tetrahydro-isocohumulone, tetrahydro-adhumulone, hexahydro-isohumulone,hexahydro-isocohumulone, and hexahydro-adhumulone.

In a further embodiment, the invention provides a method of inhibitingan inflammatory response selectively in target cells, the methodcomprising contacting the cells with a fraction isolated or derived fromhops and a second component selected from the group consisting ofrosemary, an extract derived from rosemary, a compound derived fromrosemary, a triterpene species, a diterpene lactone, and tryptanthrin.In such a method, the fraction isolated or derived from hops can beselected from the group consisting of alpha acids, isoalpha acids,reduced isoalpha acids, tetra-hydroisoalpha acids, hexa-hydroisoalphaacids, beta acids, and spent hops.

In such a method, the fraction isolated or derived from hops cancomprise a compound of a supragenus having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl;    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; and    -   wherein R, T, X, and Z are independently selected from the group        consisting of H, F, Cl, Br, I, and π orbital, with the proviso        that if one of R, T, X, or Z is a π orbital, then the adjacent        R, T, X, or Z is also a π orbital, thereby forming a double        bond.

In another embodiment of such a method, the fraction isolated or derivedfrom hops can comprise a compound of Genus A having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In still another embodiment of such a method, the fraction isolated orderived from hops can comprise a compound of Genus B having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In such a method of the invention, the fraction isolated or derived fromhops can comprise a compound selected from the group consisting ofhumulone, cohumulone, adhumulone, isohumulone, isocohumulone,isoadhumulone, dihydro-isohumulone, dihydro-isocohumulone,dihydro-adhumulone, tetrahydro-isohumulone, tetrahydro-isocohumulone,tetrahydro-adhumulone, hexahydro-isohumulone, hexahydro-isocohumulone,and hexahydro-adhumulone.

In yet another embodiment, the invention provides a method of modulatingthe inflammatory response in cells, the method comprising contacting thecells with a composition comprising a fraction isolated or derived fromhops. In an additional embodiment, the invention provides a method oftreating or inhibiting a pathological condition in a mammal associatedwith tissue-specific activation of inflammation, the method comprisingadministering to the mammal a composition comprising a fraction derivedfrom hops. In such a method, the fraction derived from hops can beselected from the group consisting of isoalpha acids, reduced isoalphaacids, tetra-hydroisoalpha acids, hexa-hydroisoalpha acids, beta acids,and spent hops.

In an embodiment of such a method, the fraction derived from hops cancomprise a compound of a supragenus having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl;    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; and    -   wherein R, T, X, and Z are independently selected from the group        consisting of H, F, Cl, Br, I, and π orbital, with the proviso        that if one of R, T, X, or Z is a π orbital, then the adjacent        R, T, X, or Z is also a π orbital, thereby forming a double        bond.

In another embodiment of such a method of the invention, the fractionderived from hops can comprise a compound of Genus A having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In addition in such a method, the fraction derived from hops cancomprise a compound of Genus B having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In another embodiment of such a method of the invention, the fractionderived from hops can comprise a compound selected from the groupconsisting of cohumulone, adhumulone, isohumulone, isocohumulone,isoadhumulone, dihydro-isohumulone, dihydro-isocohumulone,dihydro-adhumulone, tetrahydro-isohumulone, tetrahydro-isocohumulone,tetrahydro-adhumulone, hexahydro-isohumulone, hexahydro-isocohumulone,and hexahydro-adhumulone. In a particular embodiment of the method, thecomposition can comprise about 0.5 to 10000 mg or about 50 to 7500 mg ofthe fraction derived from hops. In addition, the composition cancomprise about 0.001 to 10 weight percent or about 0.1 to 1 weightpercent of the fraction is derived from hops.

In such a method of the invention, the pathological condition can beselected from the group consisting of autoimmune diseases, inflammatorydiseases, neurological diseases, and cancer. In another embodiment, thepathological condition can be selected from the group consisting ofinflammation, inflammation-associated disorders, arthritis, asthma,bronchitis, menstrual cramps, tendonitis, bursitis, skin-relatedconditions, gastrointestinal conditions, cancer, ophthalmic diseases,pulmonary inflammation, nervous system disorders, allergic rhinitis,respiratory distress syndrome, endotoxin shock syndrome,atherosclerosis, and central nervous damage.

In still another embodiment, the invention provides a method ofmodulating the amount of cyclooxygenase-2 (COX-2) activity in targetcells without substantially modulating COX-2 activity in non-targetcells, the method comprising contacting the cells with a fractionderived from hops. In such a method of the invention, the non-targetcells can also be contacted with a fraction derived from hops. Thecontacting step can be performed in vivo. In the method of theinvention, the COX-2 activity can be modulated by inhibition of COX-2gene.

In yet another embodiment, the invention provides a method of treatingor inhibiting a pathological condition in a mammal involving inhibitinginducibility or activity of cyclooxygenase-2 (COX-2), the methodcomprising administering to the mammal a composition comprising afraction derived from hops. In such a method, the fraction derived fromhops can be selected from the group consisting of isoalpha acids,reduced isoalpha acids, tetra-hydroisoalpha acids, hexa-hydroisoalphaacids, beta acids, and spent hops.

In another embodiment of the method, the fraction derived from hops cancomprise a compound of a supragenus having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl;    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; and    -   wherein R, T, X, and Z are independently selected from the group        consisting of H, F, Cl, Br, I, and π orbital, with the proviso        that if one of R, T, X, or Z is a π orbital, then the adjacent        R, T, X, or Z is also a π orbital, thereby forming a double        bond.

In such a method, the fraction derived from hops can comprise a compoundof Genus A having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In another embodiment of the method, the fraction derived from hops cancomprise a compound of Genus B having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In still another embodiment of the method, the fraction derived fromhops can comprise a compound selected from the group consisting ofcohumulone, adhumulone, isohumulone, isocohumulone, isoadhumulone,dihydro-isohumulone, dihydro-isocohumulone, dihydro-adhumulone,tetrahydro-isohumulone, tetrahydro-isocohumulone, tetrahydro-adhumulone,hexahydro-isohumulone, hexahydro-isocohumulone, andhexahydro-adhumulone.

In such a method of the invention, the pathological condition can beselected from the group consisting of wherein the pathological conditionis selected from the group consisting of inflammation,inflammation-associated disorders, arthritis, asthma, bronchitis,menstrual cramps, tendonitis, bursitis, skin-related conditions,gastrointestinal conditions, cancer, ophthalmic diseases, pulmonaryinflammation, nervous system disorders, allergic rhinitis, respiratorydistress syndrome, endotoxin shock syndrome, atherosclerosis, andcentral nervous damage.

Moreover, the invention provides a method of inhibiting prostaglandinsynthesis selectively in target cells, the method comprising contactingthe cells with a fraction derived from hops. In such a method, thefraction derived from hops can be selected from the group consisting ofisoalpha acids, reduced isoalpha acids, tetra-hydroisoalpha acids,hexa-hydroisoalpha acids, beta acids, and spent hops.

In another embodiment of the method, the fraction derived from hops cancomprise a compound of a supragenus having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl;    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; and    -   wherein R, T, X, and Z are independently selected from the group        consisting of H, F, Cl, Br, L and π orbital, with the proviso        that if one of R, T, X, or Z is a π orbital, then the adjacent        R, T, X, or Z is also a π orbital, thereby forming a double        bond.

In still another embodiment of the method, the fraction derived fromhops comprises a compound of Genus A having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In yet another embodiment of the method, the fraction derived from hopscan comprise a compound of Genus B having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In a further embodiment of the method, the fraction derived from hopscan comprise a compound selected from the group consisting ofcohumulone, adhumulone, isohumulone, isocohumulone, isoadhumulone,dihydro-isohumulone, dihydro-isocohumulone, dihydro-adhumulone,tetrahydro-isohumulone, tetrahydro-isocohumulone, tetrahydro-adhumulone,hexahydro-isohumulone, hexahydro-isocohumulone, andhexahydro-adhumulone.

The invention further provides a method of modulating NF-κB in cells notassociated with bone resorption, the method comprising contacting thecells with a composition comprising a fraction isolated or derived fromhops. The invention additionally provides a method of treating orinhibiting a pathological condition other than osteoporosis in a mammalassociated with tissue-specific activation of NF-κB, the methodcomprising administering to the mammal a composition comprising afraction isolated or derived from hops. In such a method, the fractioncan be derived from hops and selected from the group consisting ofisoalpha acids, reduced isoalpha acids, tetra-hydroisoalpha acids,hexa-hydroisoalpha acids, beta acids, and spent hops.

In another embodiment of the method, the fraction can be derived fromhops and can comprise a compound of a supragenus having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl;    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; and    -   wherein R, T, X and Z are independently selected from the group        consisting of H, F, Cl, Br, I, and π orbital, with the proviso        that if one of R, T, X, or Z is a π orbital, then the adjacent        R, T, X, or Z is also a π orbital, thereby forming a double        bond.

In another embodiment of the method, the fraction is derived from hopsand comprises a compound of Genus A having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In still another embodiment of the method, the fraction is derived fromhops and comprises a compound of Genus B having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In yet another embodiment of the method, the fraction is derived fromhops and comprises a compound selected from the group consisting ofcohumulone, adhumulone, isohumulone, isocohumulone, isoadhumulone,dihydro-isohumulone, dihydro-isocohumulone, dihydro-adhumulone,tetrahydro-isohumulone, tetrahydro-isocohumulone, tetrahydro-adhumulone,hexahydro-isohumulone, hexahydro-isocohumulone, andhexahydro-adhumulone.

In a particular embodiment of the method, the composition used in amethod of the invention can comprise about 0.5 to 10000 mg or about 50to 7500 mg of the fraction isolated or derived from hops. Also, thecomposition can comprise about 0.001 to 10 weight percent or about 0.1to 1 weight percent of the fraction isolated or derived from hops.

In a method of the invention modulating NFκB, the pathological conditioncan be selected from the group consisting of autoimmune diseases,inflammatory diseases, neurological diseases, cardiovascular diseases,and cancer. Also, the pathological condition in a method of modulatingNFκB can be selected from the group consisting of asthma, HIV-1replication, cold, and flu.

In another embodiment, the invention provides a method of modulating theamount of cyclooxygenase-2 (COX-2) activity in target cells notassociated with bone resorption without substantially modulating COX-2activity in non-target cells, the method comprising contacting the cellswith a fraction isolated or derived from hops. In the method, thenon-target cells can also be contacted with a fraction isolated orderived from hops. The contacting step can be performed in vivo. In themethod, the COX-2 activity can be modulated by inhibition of the COX-2gene.

Additionally, the invention provides a method of treating or inhibitinga pathological condition other than osteoporosis in a mammal involvinginhibiting inducibility or activity of cyclooxygenase-2 (COX-2), themethod comprising administering to the mammal a composition comprising afraction isolated or derived from hops. In a particular embodiment, thefraction can be derived from hops and selected from the group consistingof isoalpha acids, reduced isoalpha acids, tetra-hydroisoalpha acids,hexa-hydroisoalpha acids, beta acids, and spent hops.

In another embodiment of the method, the fraction is derived from hopsand comprises a compound of a supragenus having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl;    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; and    -   wherein R, T, X, and Z are independently selected from the group        consisting of H, F, Cl, Br, I, and a π orbital, with the proviso        that if one of R, T, X, or Z is a π orbital, then the adjacent        R, T, X, or Z is also a π orbital, thereby forming a double        bond.

In still another embodiment of the method, the fraction is derived fromhops and comprises a compound of Genus A having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In yet another embodiment of the method, the fraction can be derivedfrom hops and comprise a compound of Genus B having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In a particular embodiment of the method, the fraction can be derivedfrom hops and can comprise a compound selected from the group consistingof cohumulone, adhumulone, isohumulone, isocohumulone, isoadhumulone,dihydro-isohumulone, dihydro-isocohumulone, dihydro-adhumulone,tetrahydro-isohumulone, tetrahydro-isocohumulone, tetrahydro-adhumulone,hexahydro-isohumulone, hexahydro-isocohumulone, andhexahydro-adhumulone.

In another embodiment of the method, the pathological condition can beselected from the group consisting of inflammation,inflammation-associated disorders, arthritis, asthma, bronchitis,menstrual cramps, tendonitis, bursitis, skin-related conditions,gastrointestinal conditions, cancer, ophthalmic diseases, pulmonaryinflammation, nervous system disorders, allergic rhinitis, respiratorydistress syndrome, endotoxin shock syndrome, atherosclerosis, andcentral nervous damage.

Also, the invention provides a method of inhibiting prostaglandinsynthesis selectively in target cells, the method comprising contactingthe cells with a fraction derived from hops. In such a method, thefraction derived from hops can be selected from the group consisting ofisoalpha acids, reduced isoalpha acids, tetra-hydroisoalpha acids,hexa-hydroisoalpha acids, beta acids, and spent hops.

In such a method, the fraction derived from hops can comprise a compoundof a supragenus having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl;    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; and    -   wherein R, T, X, and Z are independently selected from the group        consisting of H, F, Cl, Br, I, and π orbital, with the proviso        that if one of R, T, X, or Z is a π orbital, then the adjacent        R, T, X, or Z is also a π orbital, thereby forming a double        bond.

In another embodiment of the method, the fraction derived from hops cancomprise a compound of Genus A having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In yet another embodiment of the method, the fraction derived from hopscomprises a compound of Genus B having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In still another embodiment of the method, the fraction derived fromhops can comprise a compound selected from the group consisting ofcohumulone, adhumulone, isohumulone, isocohumulone, isoadhumulone,dihydro-isohumulone, dihydro-isocohumulone, dihydro-adhumulone,tetrahydro-isohumulone, tetrahydro-isocohumulone, tetrahydro-adhumulone,hexahydro-isohumulone, hexahydro-isocohumulone, andhexahydro-adhumulone.

Moreover, the invention provides a method of inhibiting an inflammatoryresponse selectively in target cells, the method comprising contactingthe cells with a fraction derived from hops. In such a method, thefraction derived from hops can be selected from the group consisting ofisoalpha acids, reduced isoalpha acids, tetra-hydroisoalpha acids,hexa-hydroisoalpha acids, beta acids, and spent hops.

In such a method, the fraction derived from hops can comprise a compoundof a supragenus having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl;    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; and    -   wherein R, T, X, and Z are independently selected from the group        consisting of H, F, Cl, Br, L and π orbital, with the proviso        that if one of R, T, X, or Z is a π orbital, then the adjacent        R, T, X, or Z is also a π orbital, thereby forming a double        bond.

In another embodiment of the method, the fraction derived from hops cancomprise a compound of Genus A having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In still another embodiment of the method, the fraction derived fromhops can comprise a compound of Genus B having the formula:

-   -   wherein R′ is selected from the group consisting of carbonyl,        hydroxyl, OR, and OCOR, wherein R is alkyl; and    -   wherein R″ is selected from the group consisting of CH(CH₃)₂,        CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In such a method of the invention, the fraction derived from hops cancomprise a compound selected from the group consisting of cohumulone,adhumulone, isohumulone, isocohumulone, isoadhumulone,dihydro-isohumulone, dihydro-isocohumulone, dihydro-adhumulone,tetrahydro-isohumulone, tetrahydro-isocohumulone, tetrahydro-adhumulone,hexahydro-isohumulone, hexahydro-isocohumulone, andhexahydro-adhumulone. It is understood that compositions of theinvention disclosed herein can be used in the various methods of theinvention, as disclosed herein.

The invention additional provides a method of treating or inhibitingobesity in a mammal, the method comprising administering to the mammal acomposition comprising a fraction isolated or derived from hops and asecond component selected from the group consisting of rosemary, anextract derived from rosemary, a compound derived from rosemary, atriterpene species, a diterpene lactone, and tryptanthrin or othercompositions of the invention, as disclosed herein.

As disclosed herein in Examples 1, 2 and 27, the AGS gastric mucosalcell line can function as a model system for determining potentialgastrointestinal toxicity of anti-inflammatory agents. In AGS cells,COX-1 is expressed four times greater than COX-2. A lower inhibition ofPGE₂ in AGS cells is favorable because the AGS cell line expresses moreCOX-1, which maintains mucosal homeostasis. The invention thus alsoprovides a method of determining potential gastrointestinal toxicity ofan anti-inflammatory agent. The method can include the steps ofcontacting an AGS gastric mucosal cell with an anti-inflammatory agent;contacting a target inflammatory cell, for example, an A549 cell, withthe anti-inflammatory agent; determining the 50% inhibitoryconcentration (IC₅₀) of prostaglandin E₂ (PGE₂) expression for theanti-inflammatory agent in each of the AGS cell and target inflammatorycell; and determining the ratio of the IC₅₀ value of the AGS cell to theIC₅₀ value of the target inflammatory cell, wherein a ratio greater than1 indicates decreased potential gastrointestinal toxicity and a ratioless than 1 indicates increased potential gastrointestinal toxicity.

As disclosed herein, reduced isomerized alpha acids and isomerized alphaacids appear to inhibit COX-2 expression rather than directly on PGE₂(see Example 25). Further as disclosed herein, hops has no significantdose-related effect on COX-1 or COX-2 enzyme activity, supporting thathops and/or fractions isolated or derived from hops affect COX-2expression (see Example 26).

The description below is of specific examples setting forth preferredembodiments and are not intended to limit the scope.

Example 1 AGS Gastric Mucosal Cells Constitutively Express BothCyclooxygenase-1 and Cyclooxygenase-2

Summary

This example demonstrates that the AGS human gastric mucosal cell line,possessing constitutive expression of COX-1 and COX-2, has excellentpotential to serve as a model for assessing the gastrointestinaltoxicity of cyclooxygenase-inhibiting compounds.

Equipment used in this example included: an OHAS Model #E01140analytical balance, a Form a Model #F1214 biosafety cabinet (Marietta,Ohio), various pipettes to deliver 0.1 to 100 μL (VWR, Rochester, N.Y.),a cell hand tally counter (VWR Catalog #23609-102, Rochester, N.Y.), aForm a Model #F3210 CO₂ incubator (Marietta, Ohio), a hemacytometer(Hausser Model #1492, Horsham, Pa.), a Leica Model #DM IL invertedmicroscope (Wetzlar, Germany), a PURELAB Plus Water Polishing System(U.S. Filter, Lowell, Mass.), a 4° C. refrigerator (Form a Model #F3775,Marietta, Ohio), a vortex mixer (VWR Catalog #33994-306, Rochester,N.Y.), and a 37° C. water bath (Shel Lab Model #1203, Cornelius, Oreg.).

Chemicals and Reagents

Prostaglandin E₂ EIA kit Monoclonal was purchased from Cayman Chemical(Ann Arbor, Mich.). Anti-COX-1 and anti-COX-2 rabbit polygonal antiserawere obtained from Upstate Biotechnology (CITY, N.Y.); donkey anti-goatIgG-HRP was procured from Santa Cruz Biotechnology (City, Calif.). Heatinactivated Fetal Bovine Serum (FBS-HI Cat. #35-011CV), and Dulbeco'sModification of Eagle's Medium (DMEM Cat #10-013CV) was purchased fromMediatech (Herndon, Va.). All standard reagents were obtained from Sigma(St. Louis, Mo.) and were the purest commercially available.

Cell Culture

The human gastric mucosal cell line AGS was obtained from the AmericanType Culture Collection (ATCC number CRL-1739; Manassas, Va.) andsub-cultured according to the instructions of the supplier. The cellswere routinely cultured at 37° C. with 5% CO₂ in RPMI 1640 containing10% FBS, with 50 units penicillin/mL, 50 μg streptomycin/mL, 5% sodiumpyruvate, and 5% L-glutamine. Exponentially growing cells were seededinto 6-well plates and grown to confluence. A 20 μL aliquot of thesupernatant media was sampled for determination of PGE₂ content. Cellswere then washed in PBS, scraped and lysed for immunoblotting.

Protein Assay

Protein concentrations of cell lysates were determined using theNanoOrange Protein Quantitation Kit with bovine serum albumin as thestandard (Molecular Probes, Eugene, Oreg.) according to the proceduresupplied by the manufacturer. Fluorescence was determined using aPackard FluoroCount, Model BF 10000 fluorometer with the excitationfilter set at 485 nm and emission filter set at 570 nm using PackardPlateReader version 3.0 software. The I-Smart program provided with thePackard PlateReader was used to calculate the protein concentration.

Immunoblotting

Western blotting of COX-1 and COX-2 was performed using PAGEr™ GoldPrecast Gels (Bio Whittaker Molecular Applications (Rockland, Me.). AGScell lysates containing approximately 60 μg protein were loaded withLaemmli Sample Buffer into the wells of the gel in a total volume of 30μL. The vertical minigel electrophoresis chambers were made by SavantInstruments Inc. (Holbrook, N.Y.), model MV 120. Gels were run at 40mA/plate (constant current) at room temperature until the bromophenolblue stain reached the bottom of the gel, about one h. Gels were thenblotted on the polyvinyl fluoride transfer membranes (Pall Corporation,Ann Arbor, Mich.), overnight, at 500 mA and 4° C. Precision ProteinStandard molecular weight markers, unstained, broad range (BioRad,Hercules, Calif.) were used. The BioWest™ Extended durationchemiluminescent substrate, a non-isotopic, horseradish peroxidasesubstrate kit for Western blot detection (BioImaging Systems, Upland,Calif.) was used for protein visualization. Images of western blots wereacquired using a UVP Epi Chemi II Darkroom (BioImaging Systems),analyzed and enhanced by LabWorks™ Image Acquisition and AnalysisSoftware (BioImaging Systems).

PGE₂ Assay

A commercial, non-radioactive procedure for quantification of PGE₂ wasemployed (Caymen Chemical, Ann Arbor, Mich.) and the recommendedprocedure of the manufacturer was used without modification. Briefly, 25μL of the medium, along with a serial dilution of PGE₂ standard samples,were mixed with appropriate amounts of acetylcholinesterase-labeledtracer and PGE₂ antiserum, and incubated at room temperature for 18 h.After the wells were emptied and rinsed with wash buffer, 200 μL ofEllman's reagent containing substrate for acetylcholinesterase wereadded. The reaction was carried out on a slow shaker at room temperaturefor 1 h and the absorbance at 415 nm was determined. The PGE₂concentration was represented as picograms per 10⁵ cells.

Results

As seen in FIG. 6, the AGS cell line constitutively expresses both COX-1and COX-2, with COX-1 expression approximately 4-times greater thanCOX-2 expression. PGE₂ synthesis in AGS cells over 18 h was 660 pg/10⁵cells. Thus, this example demonstrates that the AGS human gastricmucosal cell line, possessing constitutive expression of COX-1 andCOX-2, has excellent potential to serve as a model for assessing thegastrointestinal toxicity of cyclooxygenase-inhibiting compounds.

In the past, the classical COX-2 hypothesis has downplayed the role ofCOX-2 expression in the gastrointestinal mucosa. While in normal gastricmucosa COX-1 is the predominant COX isozyme, as demonstrated in thisexample and in the literature, there is increasing evidence thatdetectable amount of COX-2 mRNA and protein are both constitutivelyexpressed and inducible in specific locations of the gastric mucosa inboth animals and humans [Halter, F., et al. (2001) Cyclooxygenase2-implications on maintenance of gastric mucosal integrity and ulcerhealing: controversial issues and perspectives. Gut 49, 443-453]. Recentstudies in rats have shown that whereas selective inhibition of COX-1 orCOX-2 is not ulcerogenic, combined inhibition of both COX-1 and COX-2induces severe lesions in the stomach and small intestine comparablewith the effects of NSAID such as indomethacin. This observationsuggests an important contribution of COX-2 to the maintenance ofgastrointestinal mucosal integrity.

Example 2 Inhibition of PGE₂ Synthesis in Gastric Mucosal Cells byNonsteroidal Anti-Inflammatory Drugs

Summary

This example illustrates that inhibition of PGE₂ synthesis in AGSgastric cells by NSAIDs correlates with their observed clinical gastricirritation.

Chemicals

Rofecoxib and celexocib were obtained. Diisofluorophosphate (DIFP),nimensulide, ibuprofen, salicylic acid, aspirin, indomethacin andacetaminophen were purchased from Sigma (St Louis, Mo.). All otherchemicals were obtained from suppliers as described in Example 1.

Cells

A549 (human pulmonary epithelial; ATCC number CCL-185) and AGS cells(human gastric mucosa; ATCC number CRL-1739) were obtained from theAmerican Type Culture Collection (Manassas, Va.) and sub-culturedaccording to the instructions of the supplier. The cells were routinelycultured at 37° C. with 5% CO₂ in RPMI 1640 containing 10% FBS, with 50units penicillin/mL, 50 μg streptomycin/mL, 5% sodium pyruvate, and 5%L-glutamine. On the day of the experiments, exponentially growing cellswere harvested and washed with serum-free RPMI 1640.

The log phase A549 and AGS cells were plated at 8×10⁴ cells per well in0.2 mL growth medium per well in a 96-well tissue culture plate. For thedetermination of PGE₂ inhibition by the test compounds in A549 cells,the procedure of Warner et al., also known as the WHMA-COX-2 protocolWarner, T. D., et al. (1999) Nonsteroid drug selectivities forcyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated withhuman gastrointestinal toxicity: a full in vitro analysis. Proc NatlAcad Sci USA 96, 7563-7568.] was followed with no modifications.Briefly, 24 hours after plating of the A549 cells, interleukin-1β (10ng/mL) was added to induce the expression of COX-2. After 24 hr, thecells were washed with serum-free RPMI 1640 and the test materials,dissolved in DMSO and serum-free RPMI, were added to the wells toachieve final concentrations of 25, 5.0, 0.5 and 0.05 μg/mL. Eachconcentration was run in duplicate. DMSO was added to the control wellsin an equal volume to that contained in the test wells. Sixty minuteslater, A23187 (50 μM) was added to the wells to release arachidonicacid. Twenty-five μL of media were sampled from the wells 30 minuteslater for PGE₂ determination.

Non-stimulated AGS cells were used in these studies. Twenty-four hoursafter plating in the 96-well microtiter plates, the cells were washedwith serum-free RPMI 1640 and the test materials, dissolved in DMSO andserum-free RPMI, were added to the wells to achieve final concentrationsof 25, 5.0, 0.5 and 0.05 μg/mL. Each concentration was run in duplicate.DMSO was added to the control wells in an equal volume to that containedin the test wells. Sixty minutes later, arachidonic acid was added tothe wells to achieve a final concentration of 100 μM. Twenty-five μL ofmedia were sampled from the wells 30 minutes after the addition ofarachidonic acid for PGE₂ determination.

Cell Viability

Cell viability was assessed by a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-basedcolorimetric assay (Sigma, St. Louis, Mo.). The MTT solution was addeddirectly to the wells after sampling for PGE₂ determination. Theabsorbance of each well was read at 580 nm using an ELISA plate reader.No toxicity was observed at the highest concentrations tested for any ofthe compounds.

Calculations

The median inhibitory concentration (IC₅₀) for PGE₂ synthesis wascalculated using CalcuSyn (BIOSOFT, Ferguson, Mo.). This statisticalpackage performs multiple drug dose-effect calculations using the medianeffect methods described by T-C Chou and P. Talaly [(1984) Quantitativeanalysis of dose-effect relationships: the combined effects of multipledrugs or enzyme inhibitors. Adv Enzyme Regul 22, 27-55.] herebyincorporated by reference.

Briefly, the analysis correlates the “Dose” and the “Effect” in thesimplest possible form: fa/fu=(C/C_(m))^(m), where C is theconcentration or dose of the compound and Cm is the median-effectivedose signifying the potency. Cm is determined from the x-intercept ofthe median-effect plot. The fraction affected by the concentration ofthe test material is fa and the fraction unaffected by the concentrationis fu (fu=1−fa). The exponent m is the parameter signifying thesigmoidicity or shape of the dose-effect curve. It is estimated by theslope of the median-effect plot.

The median-effect plot is a graph of x=log(C) vs y=log(fa/fu) and isbased on the logarithmic form of Chou's median-effect equation. Thegoodness of fit for the data to the median-effect equation isrepresented by the linear correlation coefficient r of the median-effectplot. Usually, the experimental data from enzyme or receptor systemshave an r>0.96, from tissue culture an r>0.90 and from animal systems anr>0.85. In the cell-based studies reported here, all linear correlationcoefficients were greater than 0.90. Experiments were repeated threetimes on three different dates. The percent inhibition at each dose wasaveraged over the three independent experiments and used to calculatethe median inhibitory concentrations reported.

Results

The highly specific COX-2 inhibitor diisofluorophosphate exhibited amedian inhibitory concentration in A549 cells of 1.19 μg/mL and did notinhibit PGE₂ synthesis in AGS cells at the highest concentration testedof 25 μg/mL (Table 3). Rofecoxib, and celexocib, selective COX-2 drugs,were 27-, and 14-times, respectively, more potent inhibitors of PGE₂synthesis in the target A549 cells than in the non-target AGS gastricmucosal cells. This finding demonstrates not only COX-2 selectivity, butalso target-tissue selectivity consistent with their lowgastrointestinal toxicity. Nimensulide, another new, selective COX-2inhibitor was equally as potent in the inhibition of PGE₂ synthesis inboth cell lines. The anti-inflammatory agent acetaminophen, purported toinhibit an unidentified isozyme of COX (COX-3) and having lowgastrointestinal toxicity, inhibited PGE₂ biosynthesis in A549 cells buthad no effect on PGE₂ synthesis in AGS gastric mucosal cells.

Alternatively and consistent with their demonstrated clinical gastrictoxicity, ibuprofen, aspirin and indomethacin all exhibited moreinhibition of PGE₂ synthesis in the AGS cell line than in the targetA549 cells. Salicylic acid, an anti-inflammatory agent that inhibits theexpression of COX-2 with little gastric irritation, was inactive in bothcell models.

TABLE 3 Median inhibitory concentrations for test comoounds in the A549and AGS cell lines. IC₅₀ A549 IC₅₀ AGS IC₅₀ AGS/ Compound [μg/mL][μg/mL] IC₅₀ A549 Diisofluorophosphate 1.19 >25 >21 Rofecoxib 0.081 2.2127.3 Celexocib 0.004 0.055 13.8 Nimensulide 0.10 0.11 1.0 Ibuprofen 0.100.05 0.50 Aspirin 0.48 0.09 0.19 Indomethacin 0.033 0.002 0.002Salicylic acid >25 >25 >1 Acetaminophen 0.607 >25 >41

These results validate the use of the AGS gastric mucosal cell line toevaluate potential gastrointestinal toxicity of anti-inflammatory agentscapable of inhibiting the synthesis of PGE₂. They also demonstratecellular specificity in the action of COX-inhibiting compounds. A ratioof 1 for IC₅₀ AGS/IC₅₀ A549 indicates IC₅₀s that are the same for boththe AGS cell and A549 cells. If the ratio is higher than 1 for IC₅₀AGS/IC₅₀ A549, then the inhibition of PGE₂ is lower for the AGS cells. Alower inhibition of PGE₂ in AGS cells is favorable because AGS cell lineexpresses more COX-1, which maintains mucosal homeostasis.

Example 3 Inhibition of PGE₂ Synthesis in Stimulated and NonstimulatedMurine Macrophages by Hops (Humulus lupulus) Compounds and Deriviatives

Summary

This example illustrates the potency of hops fractions and derivativesto inhibit COX-2 synthesis of PGE₂ preferentially over COX-1 synthesisof PGE₂ in the murine macrophage model.

Chemicals and Reagents

Bacterial lipopolysaccharide (LPS; B E. coli 055:B5) was from Sigma (St.Louis, Mo.). Hops fractions (1) alpha hop (1% alpha acids; AA), (2)aromahop OE (10% beta acids and 2% isomerized alpha acids, (3) isohop(isomerized alpha acids; IAA), (4) beta acid solution (beta acids BA),(5) hexahop gold (hexahydro isomerized alpha acids; HHIAA), (6) redihop(reduced isomerized-alpha acids; RIAA), (7) tetrahop(tetrahydro-iso-alpha acids THIAA) and (8) spent hops were obtained fromBetatech Hops Products (Washington, D.C., U.S.A.). The spent hops wereextracted two times with equal volumes of absolute ethanol. The ethanolwas removed by heating at 40° C. until a only thick brown residueremained. This residue was dissolved in DMSO for testing in RAW 264.7cells. Unless otherwise noted, all standard reagents were obtained fromSigma (St. Louis, Mo.) and were the purest commercially available. Allother chemicals and equipment were as described in Examples 1 and 2.

Cell Culture

RAW 264.7 cells, obtained from American Type Culture Collection (Catalog#TIB-71, Manassas, Va.), were grown in Dulbecco's Modification ofEagle's Medium (DMEM, Mediatech, Herndon, Va.) and maintained in logphase. The DMEM growth medium was made by adding 50 mL of heatinactivated FBS and 5 mL of penicillin/streptomycin to a 500 mL bottleof DMEM and storing at 4° C. The growth medium was warmed to 37° C. inwater bath before use.

On day one of the experiment, the log phase RAW 264.7 cells were platedat 8×10⁴ cells per well in 0.2 mL growth medium per well in a 96-welltissue culture plate in the morning. At the end of the day one (6 to 8 hpost plating), 100 μL of growth medium from each well were removed andreplaced with 100 μL fresh medium.

A 1.0 mg/mL stock solution of LPS, used to induce the expression ofCOX-2 in the RAW 264.7 cells, was prepared by dissolving 1.0 mg of LPSin 1 mL DMSO. It was vortexed until dissolved and stored at 4° C. Beforeuse, it was melted at room temperature or in a 37° C. water bath.

On day two of the experiment, test materials were prepared as 1000×stock in DMSO. In 1.7 mL microfuge tubes, 1 mL DMEM without FBS wasadded for test concentrations of 0.05, 0.10, 0.5, and 1.0 μg/mL. Two μLof the 1000×DMSO stock of the test material was added to the 1 mL ofmedium without FBS. The tube contained the final concentration of thetest material concentrated 2-fold and the tube placed in an incubatorfor 10 minutes to equilibrate to 37° C.

For COX-2 associated PGE₂ synthesis, 100 μL of medium were removed fromeach well of the cell plates prepared on day one and replaced with 100μL of equilibrated 2× final concentration of the test compounds. Cellswere then incubated for 90 minutes. Twenty μL of LPS were added to eachwell of cells to be stimulated to achieve a final concentration of 1 μgLPS/mL and the cells were incubated for 4 h. The cells were furtherincubated with 5 μM arachidonic acid for 15 minutes. Twenty-five μL ofsupernatant medium from each well was transferred to a clean microfugetube for the determination of PGE₂ released into the medium.

Following the LPS stimulation, the appearance of the cells was observedand viability was determined as described in Example 2. No toxicity wasobserved at the highest concentrations tested for any of the compounds.Twenty-five μL of supernatant medium from each well was transferred to aclean microfuge tube for the determination of PGE₂ released into themedium. PGE₂ was determined and reported as previously described inExample 1.

For COX-1 associated PGE₂ synthesis, 100 μL of medium were removed fromeach well of the cell plates prepared on day one and replaced with 100μL of equilibrated 2× final concentration of the test compounds. Cellswere then incubated for 90 minutes. Next, instead of LPS stimulation,the cells were incubated with 100 μM arachidonic acid for 15 minutes.Twenty-five μL of supernatant medium from each well was transferred to aclean microfuge tube for the determination of PGE₂ released into themedium. The appearance of the cells was observed and viability wasdetermined as described in Example 2. No toxicity was observed at thehighest concentrations tested for any of the compounds. Twenty-five μLof supernatant medium from each well was transferred to a cleanmicrofuge tube for the determination of PGE₂ released into the medium.PGE₂ was determined and reported as previously described in Example 1.The median inhibitory concentrations (IC₅₀) for PGE₂ synthesis from bothCOX-2 and COX-1 were calculated as described in Example 2.

TABLE 4 COX-2 and COX-1 inhibition in RAW 264.7 cells by hop fractionsand derviatives COX-2 COX-1 IC₅₀ IC₅₀ Test Material [μg/mL] [μg/mL]COX-1/COX-2 Alpha hop (AA) 0.21 6.2 30 Aromahop OE 1.6 4.1 2.6 Isohop(IAA) 0.13 18 144 Beta acids (BA) 0.54 29 54 Hexahop (HHIAA) 0.29 3.0 11Redihop (RIAA) 0.34 29 87 Tetrahop (THIAA) 0.20 4.0 21 Spent hops (EtOH)0.88 21 24

As seen in Table 4, all hops fractions and derivative selectivelyinhibited COX-2 over COX-1 in this target macrophage model. This was anovel and unexpected finding. The extent of COX-2 selectivity for thehops derivatives IAA and RIAA, respectively, 144- and 87-fold, wasunanticipated. Such high COX-2 selectivity combined with low medianinhibitory concentrations, has not been previously reported for naturalproducts from other sources.

Example 4 Hops Compounds and Derivatives are not Direct CyclooxygenaseEnzyme Inhibitors

Summary

This example illustrates that hops compounds and derivatives do notinhibit PGE₂ synthesis in A549 pulmonary epithelial cells atphysiologically relevant concentrations when tested using the WHMA-COX-2protocol.

Chemicals

Hops and hops derivatives used in this example were previously describedin Example 3. All other chemicals were obtained from suppliers asdescribed in Examples 1 and 2.

Cells

A549 (human pulmonary epithelial) Cells were obtained from the AmericanType Culture Collection (Manassas, Va.) and sub-cultured according tothe instructions of the supplier. The cells were routinely cultured at37° C. with 5% CO₂ in RPMI 1640 containing 10% FBS, with 50 unitspenicillin/mL, 50 μg streptomycin/mL, 5% sodium pyruvate, and 5%L-glutamine. On the day of the experiments, exponentially growing cellswere harvested and washed with serum-free RPMI 1640.

Log phase A549 cells were plated at 8×10⁴ cells per well with 0.2 mLgrowth medium per well in a 96-well tissue culture plate. For thedetermination of PGE₂ inhibition by the test compounds, the procedure ofWarner et al. [(1999) Nonsteroid drug selectivities forcyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated withhuman gastrointestinal toxicity: a full in vitro analysis. Proc NatlAcad Sci USA 96, 7563-7568], also known as the WHMA-COX-2 protocol wasfollowed with no modification. Briefly, 24 hours after plating of theA549 cells, interleukin-1β (10 ng/mL) was added to induce the expressionof COX-2. After 24 hr, the cells were washed with serum-free RPMI 1640and the test materials, dissolved in DMSO and serum-free RPMI were addedto the wells to achieve final concentrations of 25, 5.0, 0.5 and 0.05μg/mL. Each concentration was run in duplicate. DMSO was added to thecontrol wells in an equal volume to that contained in the test wells.Sixty minutes later, A23187 (50 μM) was added to the wells to releasearachidonic acid. Twenty-five μL of media were sampled from the wells 30minutes later for PGE₂ determination.

Cell viability was assessed as previously described in Example 2. Notoxicity was observed at the highest concentrations tested for any ofthe compounds. PGE₂ in the supernatant medium was determined andreported as previously described in Example 1.

The median inhibitory concentration (IC₅₀) for PGE₂ synthesis wascalculated as previously described in Example 2.

Results

At the doses tested, the experimental protocol failed to capture amedian effective concentration of any of the hops extracts orderivatives. Since the protocol requires the stimulation of COX-2expression prior to the addition of the test compounds, the likelyanswer to the failure of the test materials to inhibit PGE₂ synthesis isthat their mechanism of action is to inhibit the expression of the COX-2isozyme and not activity directly. While some direct inhibition can beobserved using the WHMA-COX-2 protocol, this procedure is inappropriatein evaluating the anti-inflammatory properties of hops compounds orderivatives of hops compounds.

Example 5 Lack of Inhibition of PGE₂ Synthesis in Gastric Mucosal Cellsby Hops (Humulus lupulus) Compounds and Deriviatives

Summary

This example illustrates the lack of PGE₂ inhibition by hops fractionsand in the AGS human gastric mucosal cell line implying low gastricirritancy potential of these compounds.

Chemicals and reagents were used as described in Example 3. AGS cellswere grown and used for testing hops compounds and derivatives asdescribed in Example 2. PGE₂ was determined and reported as previouslydescribed in Example 1. The median inhibitory concentrations (IC₅₀) forPGE₂ synthesis from AGS cells were calculated as described in Example 2.

TABLE 5 Lack of PGE₂, inhibition in AGS gastric mucosal cells by hopfractions and derviatives IC₅₀ AGS Test Material [μg/mL] Alpha hop(AA) >25 Aromahop OE >25 Isohop (IAA) >25 Beta acids (BA) >25 Hexahop(HHIAA) >25 Redihop (RIAA) >25 Tetrahop (THIAA) >25 Spent hops (EtOH)>25

As seen in Table 5, all hops fractions and derivatives were unable toinhibit PGE₂ synthesis by 50% or more at the highest concentrationstested in the AGS gastric mucosal cell line. Based on theanti-inflammatory potency exhibited by these fractions in targetmacrophages, this was a novel and unexpected finding.

Example 6 Inhibition of PGE₂ Synthesis by Rosemary Extract and CompoundsFound in Rosemary

Summary

This example illustrates the anti-inflammatory effect of rosemaryextract and compounds commonly found in rosemary, carnosic acid, ursolicacid and oleanolic acid in target cells and the effect of rosemaryextract and oleanolic acid on PGE₂ synthesis in gastrointestinal cells.

Equipment used, chemicals, cell handing and calculation of medianinhibitory concentrations were performed as previously described inExamples 1, 2 and 3. Carnosic acid, ursolic acid and oleanolic acid wereobtained from Sigma (St. Louis, Mo.). The rosemary extract was a hexaneextract obtained from selected leaves of Rosmarinus officinalis by mean(95%+/−3% rosemary extract) that complied with US regulation (21 CFR101-22). It was determined by HPLC analysis that the extract contained aminimum of 11% phenolic diterpenes (consisting of carnosic acid,carnosol, methyl carnosate, rosemadial, rosemarinic acid), 4.9% mincarnosic acid, and a minimum of 7.6% the sum of carnosol+carnosic acid.The carnosic acid was purchased from Sigma (St. Louis, Mo.) and theoleanolic acid (80%) was obtained from Sabinsa (121 Ethel Road West,Piscataway, N.J.).

TABLE 6 PGE₂ inhibition in RAW 264.7 and AGS cells by a rosemaryextract, carnosic acid, ursolic acid, and oleanolic acid. RAW 264.7 RAWor AGS IC₅₀ IC₅₀ COX-1/ Test Material (COX-2/COX-1)† [μg/mL] [μg/mL]COX-2 Rosemary extract (RAW/AGS) 0.51 4.0 7.8 Carnosic acid (RAW/RAW)0.50 231 470 Ursolic acid (RAW/RAW) 1.91 33 17 Oleanolic acid (RAW/RAW)1.15 19 17 Oleanolic acid (RAW/AGS) 1.15 5.0 4.3 †Indicates the celllines used to estimate inhibitor effects, respectively, on COX-2 orCOX-1 synthesis of PGE₂. In all cases, LPS-stimulated RAW 264.7 cellswere used to determine median inhibitory concentrations of COX-2mediated PGE₂ synthesis. For the estimation of the effects of testmaterials on COX-1-mediated synthesis, either non-stimulated RAW 264.7or non-stimulated AGS cells were used.

Results

All test materials exhibited potent inhibition of PGE₂ synthesis inLPS-stimulated RAW 264.7 cells indicating inhibition of the COX-2isozyme (Table 6). Surprisingly, the rosemary extract was more potentthan ursolic and oleanolic acids and equal to pure carnosic acid inpotency with a median inhibitory concentration of 0.5 μg testmaterial/mL medium. Since the rosemary extract contained only 11%carnosic acid or derivative, the inference is that the interaction ofthe carnosic acid derivatives or the myriad of other compounds in therosemary extract were acting in concert or synergistically to providesuch a potent inhibition of COX-2. Alternatively, one of the compoundspreviously identified in rosemary and listed earlier has extremely highpotency for inhibiting COX-2 mediated synthesis of PGE₂.

In non-stimulated RAW 264.7 cells, the pure compounds were relativelyinactive exhibiting IC₅₀ values of 231, 33 and 19 μg/mL, respectively,for carnosic, ursolic and oleanolic acids. This indicated a strongpreference for COX-2 inhibition over COX-1 for synthesis of PGE₂ in theRAW 264.7 target cell model. This extent of COX isozyme selectivity hasnever been reported in the literature and was an unexpected result. Inthe AGS gastric mucosal cell line, however, both the rosemary extractand oleanolic acid exhibited potent inhibition of PGE₂ synthesis.

Example 7 Synergistic Inhibition of PGE₂ Synthesis in Target Cells byHops CO₂-Extract in Combination with Triterpenoids Oleanolic Acid andUrsolic Acid

Equipment used, chemicals, cell handing and calculation of medianinhibitory concentrations were performed as previously described inExamples 1, 2 and 3. The hops CO2-extract was purchased from Hopunion,(Yakama, Wash.) and contained 30 to 60% alpha-acids and 15 to 45%beta-acids. Oleanolic and ursolic acids and were obtained from Sigma(St. Louis, Mo.) and were the highest purity commercially available(>98%).

Synergy of test components was quantified using the combination index(CI) parameter. The CI of Chou-Talaly is based on the multipledrug-effect and is derived from enzyme kinetic models (Chou, T.-C. andTalalay, P. (1977) A simple generalized equation for the analysis ofmultiple inhibitions of Michaelis-Menten kinetic systems. J. Biol. Chem.252:6438-6442). The equation determines only the additive effect ratherthan synergism or antagonism. However, we define synergism as a morethan expected additive effect, and antagonism as a less than expectedadditive effect as proposed by Cho and Talalay Using the designation ofCI=1 as the additive effect, we obtain for mutually exclusive compoundsthat have the same mode of action or for mutually non-exclusive drugsthat have totally independent modes of action the followingrelationships: CI<1, =1, and >1 indicating synergism, additivity andantagonism, respectively.

Results

The 4:1 (CO₂-extract:triterpenoid) combination tested in RAW 264.7 cellsexhibited potent synergy over the entire dose-response curve.Combination indexes computed for both test materials at the IC₅₀, IC₇₅and IC₉₀ are presented in Table 7. As described in this example, thesynergy of these combinations covered a concentration range of 0.001 to50 μg/mL of each component of the combination.

TABLE 7 Computed Combination Indexes for the dose-response curves of 1:4combinations of a CO₂-extract of hops and the triterpenes oleanolic andursolic acid Test Material CI₅₀ CI₇₅ CI₉₀ Mean CI CO₂-Extract:Oleanolicacid [1:4] 0.514 0.461 0.414 0.463 CO₂-Extract:Ursolic acid [1:4] 0.5290.650 0.806 0.662

Example 8 Synergistic Inhibition of PGE₂ Synthesis by Hops Combinationswith an Extract of Rosemary in Target and Nontarget Cells

Summary

This example illustrates synergy of combinations of reduced isomerizedalpha acids and rosemary extract on target A549 cells and synergisticantagonism of rosemary inhibition of PGE₂ synthesis in AGS gastricmucosal cells.

Equipment used, chemicals, cell handing and calculation of medianinhibitory concentrations were performed as previously described inExamples 1, 2, 3 and 4. Several differences in the protocol for testingin the A549 cells were incorporated in this example. First, testmaterials were added to the medium 60 minutes prior to stimulation withIL-1β. Second, in the determination of dose-response curves, 5 μMarachidonic acid was used in place of the calcium ionophore A23187.Synergy of the combinations was computed as described in Example 7.

Results

Table 8 shows PGE₂ inhibition by reduced isomerized alpha-acids,rosemary extract and a 2:1 combinations of reduced isomerizedalpha-acids and rosemary extract in IL-1β stimulated A549 cells. Thiscell line represents a model target cell for anti-inflammatory efficacy.Median inhibitory concentrations for reduced isomerized alpha-acids androsemary extract independently were, respectively, 0.84 and 1.3 μg/mL.The 2:1 combination of reduced isomerized alpha-acids and rosemaryextract exhibited synergy at and below the median inhibitoryconcentration of the combination.

Table 9 shows inhibition of PGE₂ synthesis in the human gastric AGScells. These cells represent a model for gastrointestinal toxicity ofprostaglandin inhibitors. Test materials exhibiting inhibition of PGE₂synthesis in these cells would be expected to demonstrate gastricirritation and ulceration with chronic use. The inhibition of PGE₂synthesis by rosemary extract was synergistically antagonized by a 2:1combination of reduced isomerized alpha-acids and rosemary extract. Thisunexpected result represents a novel finding of synergistic antagonism.

TABLE 8 Median inhibitory concentrations and combination index for PGE₂inhibition by reduced isomerized alpha-acids, rosemary extract and acombination of isomerized alpha-acids and rosemary extract inIL-1β-stimulated A549 cells IC₅₀ Combination Index < 1.0† Test Material[μg/mL] [μg/mL] Reduced isomerized alpha-acids 0.84 (RIAA) Rosemaryextract 1.3 RIAA:Rosemary 2:1 0.48 At 0.48 and below †The combinationindex was less than 1 over the portion of the dose-response curve at andbelow the IC50 value indicating synergistic inhibition of PGE₂ synthesisby the combination at these concentrations.

TABLE 9 Synergy of a 1:1 combination of reduced isomerized alpha-acidswith rosemary extract resulting in a reduction of PGE₂ inhibition in AGSgastric mucosal cells. IC₅₀ Test Material [μg/mL] Combination IndexReduced isomerized alpha-acids (RIAA) >25 — Rosemary 4.0 — RIAA:Rosemary1:1 >25 >1.0† †The combination index was greater than 1 over the entiredose-response curve indicating synergistic antagonism of PGE₂ inhibitionby the combination.

While this example only presents the combination of rosemary extractwith one of the hops derivative, reduced isomerized alpha-acids, itwould be obvious for one skilled in the art to assume to expect the sameresults with other hops derivatives that also show no PGE₂ inhibitionwith AGS cells at dose as high as 25 μg/mL. Examples of these hopsderivatives would include isomerized-alpha acids, hexahydro-isomerizedalpha acids, tetrahydro-iso-alpha acids and extracts of spent hops.

Example 9 Synergistic Inhibition of PGE₂ Synthesis by Reduced IsomerizedAlpha-Acids and Oleanolic Acid in Target Cells with no Effect on PGE₂Synthesis in Nontarget Cells

Summary

This example illustrates that reduced isomerized alpha-acids exhibitstrong synergy with the triterpene oleanolic acid in the inhibition ofPGE₂ synthesis is the target A549 cells and synergistically antagonizeoleanolic acid inhibition of PGE₂ synthesis in gastric cells.

Equipment used, chemicals, cell handing and calculation of medianinhibitory concentrations were performed as previously described inExamples 1, 2, 3 and 4. Several differences in the protocol for testingin the A549 cells were incorporated in this example. First, testmaterials were added to the medium 60 minutes prior to stimulation withIL-1β. Second, in the determination of dose-response curves, A549 cellsremained in the presence of test material overnight before the samplingof media for PGE₂ determination. Synergy of the combinations wascomputed as described in Example 7. Reduced isomerized alpha-acids wereobtained as a one percent aqueous solution from John Haas, Inc. (Yakima,Wash.) and oleanolic acid was obtained from Sabinsa (Piscataway, N.J.)and was 80% pure. Synergy of the combinations was computed as describedin Example 7.

Results

Table 10 shows PGE₂ inhibition by oleanolic acid, reduced isomerizedalpha-acids and various combinations of reduced isomerized alpha-acidsand oleanolic acid in A549 cells. This cell line represents a modeltarget cell for anti-inflammatory efficacy. Median inhibitoryconcentrations for reduced isomerized alpha-acids and oleanolic acidindependently were, respectively, 0.03 and 0.39 μg/mL. Combinations ofreduced isomerized alpha-acids and oleanolic acid consisting of 10:1,5:1, and 1:5, respectively, exhibited synergy on the dose-response curveat combined concentrations of 0.11, 0.38 and 0.76 μg/mL. Thus, when thesum of the two components was equal to or less than 0.11, 0.38 or 0.76μg/mL, their ability to inhibit PGE₂ synthesis was greater than the sumof their individual activities.

TABLE 10 Median inhibitory concentrations and combination indexes forPGE₂ inhibition by reduced isomerized alpha-acids, oleanolic acid andfour combinations of isomerized alpha- acids and oleanolic acid inIL-1β-stimulated A549 cells. IC₅₀ Test Material [μg/mL] CombinationIndex < 1.0 Oleanolic acid (80% Sabinsa) 0.390 — Reduced isomerizedalpha-acids 0.028 — (RIAA) RIAA:Oleanolic acid − [10:1] 0.042 At 0.11μg/mL and below RIAA:Oleanolic acid − [5:1] 0.059 At 0.38 μg/mL andbelow RIAA:Oleanolic acid − [1:5] 0.022 At 0.76 μg/mL and belowRIAA:Oleanolic acid − [1:10] 0.166 No † The combination index was lessthan 1 over the portion of the dose-response curve at the tabulatedvalues indicating synergistic inhibition of PGE₂ synthesis by thecombination at and below these concentrations.

Table 11 shows inhibition of PGE₂ synthesis in the human gastric AGScells. These cells represent a model for gastrointestinal toxicity ofprostaglandin inhibitors. Test materials exhibiting inhibition of PGE₂synthesis in these cells would be expected to demonstrate gastricirritation and ulceration with chronic use. The inhibition of PGE₂synthesis by oleanolic acid was synergistically antagonized by allcombinations with reduced isomerized alpha-acids. This unexpected resultrepresents a novel finding of synergistic antagonism.

TABLE 11 Synergy of reduced isomerized alpha-acids with oleanolic acidresulting in a reduction of PGE₂ inhibition in AGS gastric mucosal cellsIC₅₀ Combination Test Material [μg/mL] Index > 1.0† Oleanolic acid 5.0 —Reduced isomerized alpha-acids (RIAA >25 — RIAA:Oleanolic acid -[10:1] >25 Antagonism RIAA:Oleanolic acid - [5:1] >25 AntagonismRIAA:Oleanolic acid - [1:5] >25 Antagonism RIAA:Oleanolic acid -[1:10] >25 Antagonism †When CI > 1.0 at the IC₅₀, the combination issaid to exhibit antagonism in the inhibition of PGE₂ synthesis by AGScells.

While this example only presents the combination of oleanolic acid withone of the hops derivative, reduced isomerized alpha-acids, it would beobvious for one skilled in the art to assume to expect the same resultswith other hops derivatives that also show no PGE₂ inhibition with AGScells at dose as high as 25 μg/mL. Examples of these hops derivativeswould include isomerized-alpha acids, hexahydro-isomerized alpha acids,tetrahydro-iso-alpha acids and extracts of spent hops.

Example 10 Synergistic Inhibition of PGE₂ Synthesis by a Combination ofReduced Isomerized Alpha Acids with Tryptanthrin in Target Cells with NoEffect on PGE₂ Synthesis in Nontarget Cells

Summary

This example illustrates a potent synergy of a 1:1 combination ofreduced isomerized alpha acids and tryptanthrin on target A549 cells andsynergistic antagonism of tryptanthrin inhibition of PGE₂ synthesis inAGS gastric mucosal cells.

Equipment used, chemicals, cell handing and calculation of medianinhibitory concentrations were performed as previously described inExamples 1, 2, 3, 4 and 9. Reduced isomerized alpha-acids were obtainedas a one percent aqueous solution from John Haas, Inc. (Yakima, Wash.)and tryptanthrin was obtained from Waco Chemicals (Richmond, Va.) andwas the highest purity commercially available. Several differences inthe protocol for testing in the A549 cells were incorporated in thisexample. First, test materials were added to the medium 60 minutes priorto stimulation with IL-1β. Second, in the determination of dose-responsecurves, A549 cells remained in the presence of test material overnightbefore the sampling of media for PGE₂ determination. Synergy of thecombinations was computed as described in Example 7.

Results

Table 12 shows PGE₂ inhibition by reduced isomerized alpha-acids,tryptanthrin and a 1:1 combination of reduced isomerized alpha-acids andtryptanthrin in IL-1β stimulated A549 cells. This cell line represents amodel target cell for anti-inflammatory efficacy. Median inhibitoryconcentrations for reduced isomerized alpha-acids and tryptanthrinindependently were, respectively, 0.0.028 and 0.30 μg/mL. The 1:1combination of reduced isomerized alpha-acids and tryptanthrin exhibitedsynergy over the entire dose-response curve.

Table 13 shows inhibition of PGE₂ synthesis in the human gastric AGScells. These cells represent a model for gastrointestinal toxicity ofprostaglandin inhibitors. Test materials exhibiting inhibition of PGE₂synthesis in these cells would be expected to demonstrate gastricirritation and ulceration with chronic use. The inhibition of PGE₂synthesis by tryptanthin was synergistically antagonized by a 1:1combination of reduced isomerized alpha-acids and tryptanthrin orconjugates thereof. This unexpected result represents a novel finding ofsynergistic antagonism.

TABLE 12 Median inhibitory concentrations and combination index for PGE₂inhibition by reduced isomerized alpha-acids, tryptanthrin and acombination of isomerized alpha-acids and tryptanthrin inIL-1β-stimulated A549 cells IC₅₀ Test Material [μg/mL] Combination IndexReduced isomerized alpha-acids RIAA 0.028 — Tryptanthrin 0.300 —RIAA:Tryptanthrin - [1:1] 3.1 × 10⁷ <1.0† †The combination index wasless than 1 over the entire dose-response curve indicating synergisticinhibition of PGE₂ synthesis by the combination.

TABLE 13 Synergy of combinations of reduced isomerized alpha-acids withtryptanthrin resulting in a reduction of PGE₂ inhibition in AGS gastricmucosal cells. IC₅₀ Test Material [μg/mL] Combination Index Reducedisomerized alpha-acids (RIAA) >25 — Tryptanthrin 4.2 —RIAA:Tryptanthrin - [1:1] >25 >1.0† †The combination index was greaterthan 1 over the entire dose-response curve indicating synergisticantagonism of PGE₂ inhibition by the combination.

While this example only presents the combination of tryptanthrin withone of the hops derivative, reduced isomerized alpha-acids, it would beobvious for one skilled in the art to assume to expect the same resultswith other hops derivatives that also show no PGE₂ inhibition with AGScells at dose as high as 25 μg/mL. Examples of these hops derivativeswould include isomerized-alpha acids, hexahydro-isomerized alpha acids,tetrahydro-iso-alpha acids and extracts of spent hops.

Example 11 Ex Vivo Inhibition of PGE₂ Synthesis by a Plasma Sample froma Human Receiving a Combination Containing Hops Derivatives, a RosemaryExtract and Oleanolic Acid

Summary

This example demonstrates the presence of PGE₂ inhibiting materials in ahuman subject following ingestion of a 5:5:1 combination of reducedisomerized alpha acids, rosemary extract and oleanolic acid three timesper day for five days.

Equipment used, chemicals, RAW 264.7 cell handing and calculation ofPGE₂ concentrations were performed as previously described in Examples1, 2, and 3. Reduced isomerized alpha acids, rosemary extract andoleanolic acid were as described in Examples 3, 6 and 7, respectively.Gel caps were made to contain 200 mg reduced isomerized alpha acids, 200mg rosemary and 40 mg oleanolic acid in an oil base. Plasma samples wereobtained from a human volunteer prior to and five days after consumingthree capsules per day for five days. Capsules were taken atapproximately eight-hour intervals throughout the day. On the fifth day,blood was drawn one hour before taking the last capsule and 1, 2, 4 and7 hours after dosing. All PGE₂ assays in plasma samples were replicatedeight times. Outliers were defined and eliminated if the value was morethan three standard deviations from the group mean computed without theperceived outlier. Raw data with and without the outliers were graphed.Concentrations of test material in plasma relating to percent PGE₂inhibition were estimated using a standard curve of the combination incommercial plasma (Gibco, Grand Island, N.Y.).

FIG. 7[A] illustrates the inhibition of PGE₂ synthesis by the plasmasamples at the indicated times. A 9- to 3-fold increase in PGE₂inhibition was observed during the first post-dosing hour. Effectivehalf-life (time to reduce the ability to inhibit PGE₂ synthesis byone-half) of the test material was approximately four hours.

Estimates of test material relating to the observed percentageinhibition of PGE₂ synthesis in RAW 264.7 cells are presented in FIG.7[B]. Using only the data with outliers removed, a 12.5-fold increase intest material concentration was noted during the first hour. A maximalconcentration of 880 ng/mL plasma was seen at both the 1 and 2post-dosing hours. The concentration half-live was approximately 2.2hours. The lack of consistency between the effective half-life andconcentration half-life may be inferred to be due to the synergy ofcomponents in the formulation. Efficacy is extended due to positive andsynergistic interactions among the isomerized alpha acids, the myriad ofcompounds in the rosemary extract and oleanolic acid as has beendemonstrated by the examples in this application.

Example 12 Normalization of Joint Function Following Trauma

A representative composition of the preferred embodiments as a dietarysupplement would be in an oral formulation, i.e. tablets or gel capsthat would supply one of the following combinations: 0.1 to 10 mgisocohumulone/kg per day; 0.01 to 10 mg dihydroadhumulone/kg per day,0.01 to 10 mg tetrahydro-isocohumulone/kg per day; 0.01 to 10 mg/kg perday of hexahydro-isohumulone/kg per day for a 70 kg person.

Normalization of joint movement following physical trauma due toexercise or repetitive movement stress would be expected to occurfollowing two to ten doses. This result would be expected in allanimals.

Example 13 Normalization of Joint Function Following Trauma

A representative composition of the preferred embodiments as a dietarysupplement would be in an oral formulation, i.e. tablets or gel capsthat would supply one of the following combinations:

17 mg reduced isomerized alpha-acid/kg per day, 17 mg rosemaryextract/kg per day and 17 mg ursolic acid/kg per day;

17 mg reduced isomerized alpha-acid/kg per day, 17 mg rosemaryextract/kg per day and 3.4 mg ursolic acid/kg per day;

34 mg reduced isomerized alpha-acid/kg per day, 34 mg rosemaryextract/kg per day and 3.4 mg ursolic acid/kg per day;

340 mg reduced isomerized alpha-acid/kg per day, 340 mg rosemaryextract/kg per day and 3.4 mg ursolic acid/kg per day;

17 mg reduced isomerized alpha-acid/kg per day, 17 mg rosemaryextract/kg per day and 85 mg ursolic acid/kg per day;

17 mg reduced isomerized alpha-acid/kg per day, 17 mg rosemaryextract/kg per day and 170 mg ursolic acid/kg per day; or

17 mg reduced isomerized alpha-acid/kg per day, 17 mg rosemaryextract/kg per day and 1700 mg ursolic acid/kg per day for a 70 kgperson.

Normalization of joint movement following physical trauma due toexercise or repetitive movement stress would be expected to occurfollowing two to ten doses. This result would be expected in allanimals.

Example 14 Clinical Effectiveness of Lotion Formulations in theTreatment of Acne Rosacea

A lotion designed to contain one of the following:

-   -   1. 0.1% wt of the isomerized alpha-acid isocohumulone;    -   2. 0.1% wt of the reduced isomerized alpha-acid        dihydro-adhumulone;    -   3. 0.1% wt of the tetrahydroisoalpha-acid        tetrahydro-isocohumulone; or    -   4. 0.1% wt hexahydro-isohumulone        is applied to affected areas of patients who have exhibited acne        rosacea as diagnosed by their health practitioner and confirmed        by an independent board-certified dermatologist.

Self-evaluation tests and are administered one week prior to the studyto quantify the surface area affected and redness. In addition, similarvariables are scored by the professional clinical staff not aware of thepatients treatment status. These evaluations are repeated on Days 0, 7,14 and 21.

Patients are randomly assigned to the test formulation or placebo at thestart of the study. The test formulation and placebo are applied to theaffected area one or two times per day. Treatment for health conditionssuch as diabetes, hypertension, etc. is allowed during the study. Scoresare statistically compared between the test formulation and the placebofor each of the four observational periods. Patients treated with thecomposition of the preferred embodiments in a lotion formulation areconsidered improved if the patients' scores improve by greater than 20%from the pre-test scores within each category evaluated. The percentageof persons exhibiting improvement is compared between the combinationformulations and the placebo control. The difference between the twogroups is considered statistically significant if the probability ofrejecting the null hypothesis when true is less than five percent.

Example 15 Clinical Effectiveness of Lotion Formulations in theTreatment of Acne Rosacea

A lotion designed to contain one of the following:

-   -   1. 0.1% wt of the alpha-acid humulone;    -   2. 0.1% wt of the isomerized alpha-acid isocohumulone;    -   3. 0.1% wt of the reduced isomerized alpha-acid        dihydro-adhumulone;    -   4. 0.1% wt of the tetrahydroisoalpha-acid        tetrahydro-isocohumulone; or    -   5. 0.1% wt of the hexahydroisoalpha-acid hexahydro-isohumulone        is applied to affected areas of patients who have exhibited acne        rosacea as diagnosed by their health practitioner and confirmed        by an independent board-certified dermatologist.

Self-evaluation tests and are administered one week prior to the studyto quantify the surface area affected and redness. In addition, similarvariables are scored by the professional clinical staff not aware of thepatients treatment status. These evaluations are repeated on Days 0, 7,14 and 21.

Patients are randomly assigned to the test formulation or placebo at thestart of the study. The test formulation and placebo are applied to theaffected area one or two times per day. Treatment for health conditionssuch as diabetes, hypertension, etc. is allowed during the study. Scoresare statistically compared between the test formulation and the placebofor each of the four observational periods. Patients treated with thecomposition of the preferred embodiments in a lotion formulation areconsidered improved if the patients' scores improve by greater than 20%from the pre-test scores within each category evaluated. The percentageof persons exhibiting improvement is compared between the combinationformulations and the placebo control. The difference between the twogroups is considered statistically significant if the probability ofrejecting the null hypothesis when true is less than five percent.

Example 16 Clinical Effectiveness of Lotion Formulations in theTreatment of Acne Rosacea

A lotion designed to contain one of the following:

-   -   1. 0.1% wt of the alpha-acid humulone and 0.1% trypanthrin;    -   2. 0.1% wt of the isomerized alpha-acid isocohumulone and 0.1%        trypanthrin;    -   3. 0.1% wt of the reduced isomerized alpha-acid        dihydro-adhumulone and 0.1% tryptanthrin;    -   4. 0.1% wt of the tetrahydroisoalpha-acid        tetrahydro-isocohumulone and 0.1% tryptanthrin; or    -   5. 0.1% wt of the hexahydroisoalpha-acid hexahydro-isohumulone        and 0.1% tryptanthrin        is applied to affected areas of patients who have exhibited acne        rosacea as diagnosed by their health practitioner and confirmed        by an independent board-certified dermatologist.

Self-evaluation tests and are administered one week prior to the studyto quantify the surface area affected and redness. In addition, similarvariables are scored by the professional clinical staff not aware of thepatients treatment status. These evaluations are repeated on Days 0, 7,14 and 21.

Patients are randomly assigned to the test formulation or placebo at thestart of the study. The test formulation and placebo are applied to theaffected area one or two times per day. Treatment for health conditionssuch as diabetes, hypertension, etc. is allowed during the study. Scoresare statistically compared between the test formulation and the placebofor each of the four observational periods. Patients treated with thecomposition of the preferred embodiments in a lotion formulation areconsidered improved if the patients' scores improve by greater than 20%from the pre-test scores within each category evaluated. The percentageof persons exhibiting improvement is compared between the combinationformulations and the placebo control. The difference between the twogroups is considered statistically significant if the probability ofrejecting the null hypothesis when true is less than five percent.

Example 17 Clinical Effectiveness of a Lotion Formulation in theTreatment of Psoriasis

This example is performed in the same manner as described in Examples14, 15 and 16 except that the composition is applied to affected areasof patients who have exhibited psoriasis as diagnosed by their ownpractitioner and confirmed by an independent board-certifieddermatologist. Self-evaluation tests are administered one week prior tothe study to quantify the surface area affected and skin condition. Inaddition, similar variables are scored by the professional clinicalstaff not aware of the patients treatment status. These evaluations arerepeated on Days 0, 7, 30 and 60.

Patients are randomly assigned to the test formulation or placebo at thestart of the study. The test formulation and placebo are applied to theaffected area one or two times per day. Treatment for health conditionssuch as diabetes, hypertension, etc. is allowed during the study. Scoresare statistically compared between the test formulation and the placebofor each of the four observational periods. Patients treated with thecomposition of the preferred embodiments as the test lotion formulationare considered improved if the patients' scores improve by greater than20% from the pre-test scores within each category evaluated. Thepercentage of persons exhibiting improvement is compared between thetest formulation and the placebo control. The difference between the twogroups is considered statistically significant if the probability ofrejecting the null hypothesis when true is less than five percent.

Example 18 Clinical Effectiveness of a Formulation in the Treatment ofAlzheimer's Disease

An oral formulation as described in Examples 12 and 13 is administeredto patients who have manifested an early stage of Alzheimer's Disease(AD), as diagnosed by their practitioner and confirmed by an independentboard-certified neurologist. Two weeks before the clinical trial, thepatients undergo appropriate psychoneurological tests such as the MiniMental Status Exam (MMSE), the Alzheimer Disease Assessment Scale(ADAS), the Boston Naming Test (BNT), and the Token Test (TT).Neuropsychological tests are repeated on Day 0, 6 weeks and 3 months ofthe clinical trial. The tests are performed by neuropsychologists whoare not aware of the patient's treatment regimen.

Patients are randomly assigned to the test formulation or placebo at thestart of the study. The test formulation and placebo are taken orallyone or two times per day. Treatment for conditions such as diabetes,hypertension, etc. is allowed during the study. Scores are statisticallycompared between the test formulation and the placebo for each of thethree observational periods. Without treatment, the natural course of ADis significant deterioration in the test scores during the course of theclinical trial. Patients treated with the composition of the preferredembodiments as the test formulation are considered improved if thepatients' scores remain the same or improve during the course of theclinical trial.

Example 19 Oral Formulation in the Treatment and Prevention of ColonCancer

An oral formulation as described in Examples 12 and 13 is administeredto patients who have manifested an early stage of colon cancer asdiagnosed by their own practitioner and confirmed by a independentboard-certified oncologist.

Patients are randomly assigned to the test formulation or a placebo atthe start of the study. The test formulation and placebo are takenorally one or two times per day. Treatment for conditions such asdiabetes, hypertension, etc. is allowed during the study. Endoscopicevaluations are made at one, two, six and twelve months. Evidence ofreappearance of the tumor during any one of the four follow-up clinicalvisits is considered a treatment failure. The percentage of treatmentfailures is compared between the test formulation and the placebocontrol. Under the experimental conditions described, the test materialis expected to decrease the tumor incidence with respect to the controlgroup. The difference between the two groups is considered statisticallysignificant if the probability of rejecting the null hypothesis whentrue is less than five percent.

Example 20 Oral Formulation for the Treatment of Irritable BowelSyndrome

An oral formulation as described in Examples 12 and 13 is administeredto patients who have manifested irritable bowel syndrome as diagnosed bytheir practitioner. Normal bowel functioning is restored within 48hours.

Example 21 Normalization of Joint Functioning in Osteoarthritis

Using compositions described in Examples 12 and 13 normalization ofjoint stiffness due to osteoarthritis occurs following five to twentydoses, in the presence or absence of glucosamine or chondroitin sulfate.In addition, the composition does not interfere with the normal jointrebuilding effects of these two proteoglycan constituents, unliketraditional non-steroidal anti-inflammatory agents.

Example 22 Mite Dust Allergens Activate PGE₂ Biosynthesis in A549Pulmonary Cells

Summary

This example illustrates that house mite dust allergens can induce PGE₂biosynthesis in pulmonary epithelial cells.

Background

Sensitivity to allergens is a problem for an increasing number ofconsumers. This issue has been complicated by a surprising increase inasthma over the past few years. Asthma suffers are especially sensitiveto airborne allergens. Allergy rates are also on the rise. This givesrise to increased awareness of the causes of allergy symptoms and how todecrease the associated discomfort. Approximately 10% of the populationbecome hypersensitized (allergic) upon exposure to antigens from avariety of environmental sources. Those antigens that induce immediateand/or delayed types of hypersensitivity are known as allergens. Theseinclude products of grasses, trees, weeds, animal dander, insects, food,drugs, and chemicals. Genetic predisposition of an individual isbelieved to play a role in the development of immediate allergicresponses such as atopy and anaphylaxis whose symptoms include hayfever, asthma, and hives.

Many allergens are protein-based molecules, and these protein allergenscan originate from many sources. It has been know for some time that oneof the most common sources of allergens in a house is from dust mites.Of course, as is the case with all allergens, only certain people areallergic to dust mite allergens. But this group of people can be quitelarge in many areas, especially in hot humid areas. For example, in thesoutheastern United States of America, where it is both hot and humidfor much of the year, the incidence of house dust mite allergies in thegeneral population can be as high as 25%. House dust mites thrive inplush carpets, overstuffed upholstery, cushy bed comforters and thelike.

Methods

Mite Dust Allergen Isolation

Dermatophagoides farinae are the American house dust mite. D. farinaewere cultured on a 1:1 ratio of Purina Laboratory Chow (Ralston Purina,Co, St. Louis, Mo.) and Fleischmann's granulated dry yeast (StandardBrands, Inc. New York, N.Y.) at room temperature and 75% humidity. Livemites were aspirated from the culture container as they migrated fromthe medium, killed by freezing, desiccated and stored at 0% humidity.The allergenic component of the mite dust was extracted with water atambient temperature. Five-hundred mg of mite powder were added to 5 mLof water (1:10 w/v) in a 15 mL conical centrifuge tube (VWR, Rochester,N.Y.), shaken for one minute and allowed to stand overnight at ambienttemperature. The next day, the aqueous phase was filtered using a 0.2 μmdisposable syringe filter (Nalgene, Rochester, N.Y.). The filtrate wastermed mite dust allergen and used to test for induction of PGE₂biosynthesis in A549 pulmonary epithelial cells.

Cell Culture and Treatment

This experiment involved the human airway epithelial cell line, A549(American Type Culture Collection, Bethesda, Md.). The cells werecultured and treated as previously described in Example 2. Mite allergenwas added to the culture medium to achieve a final concentration of 1000ng/mL. Twenty-four hours later, the culture medium was sampled for PGE₂concentration.

PGE₂ Assay

Determination of PGE₂ in the culture medium was performed as previouslydescribed in Example 1.

Statistical Analysis

Means of eight replicates per treatment were computed using Excel®spreadsheets (Microsoft, Redmond, Wash.).

Results

Mite allergen treatment increased PGE₂ biosynthesis 6-fold in A549 cellsrelative to the solvent treated controls (FIG. 8).

Example 23 Hops Derivatives Inhibit Mite Dust Allergen Activation ofPGE₂ Biosynthesis in A549 Pulmonary Cells

Summary

This example illustrates that hops derivatives are capable of inhibitingthe PGE₂ stimulatory effects of mite dust allergens in A549 pulmonarycells.

Methods

The cell line and testing procedures are as described in Example 22. Inaddition to mite dust allergen, test materials included Hops fractions(1) alpha hop (1% alpha acids; AA), (2) aromahop OE (10% beta acids and2% isomerized alpha acids, (3) isohop (isomerized alpha acids; IAA), (4)beta acid solution (beta acids BA), (5) hexahop gold (hexahydroisomerized alpha acids; HHIAA), (6) redihop (reduced isomerized-alphaacids; RIAA), and (7) tetrahop (tetrahydro-iso-alpha acids (THIAA). Testmaterials at a final concentration of 10 μg/mL were added 60 minutesprior to the addition of the mite dust allergen.

Results

Table 15 depicts the extent of inhibition of PGE₂ biosynthesis by hopsderivatives in A549 pulmonary cells stimulated by mite dust allergen.All hops derivatives were capable of significantly inhibiting thestimulatory effects of mite dust allergens.

TABLE 15 PGE₂ inhibition by hops derviatives in A549 pulmonaryepithelial cells stimulated by mite dust allergen Percent Inhibition ofTest Material PGE₂ Biosynthesis Alpha hop (AA) 81 Aromahop OE 84 Isohop(IAA) 78 Beta acids (BA) 83 Hexahop (HHIAA) 82 Redihop (RIAA) 81Tetrahop (THIAA) 76

In conclusion, it would also be useful to identify a natural formulationof compounds that would inhibit expression of COX-2, inhibitprostaglandin synthesis selectively in target cells, or inhibitinflammation response selectively in target cells.

A preferred embodiment comprises compositions containing at least onefraction isolated or derived from hops (Humulus lupulus). Examples offractions isolated or derived from hops are alpha acids, isoalpha acids,reduced isoalpha acids, tetra-hydroisoalpha acids, hexa-hydroisoalphaacids, beta acids, and spent hops. Preferred compounds of fractionsisolated or derived from hops, include, but are not limited to,humulone, cohumulone, adhumulone, isohumulone, isocohumulone,isoadhumulone, dihydro-isohumulone, dihydro-isocohumulone,dihydro-adhumulone, tetrahydro-isohumulone, tetrahydro-isocohumulone,tetrahydro-adhumulone, hexahydro-isohumulone, hexahydro-isocohumulone,and hexahydro-adhumulone. Preferred compounds can also bearsubstituents, such as halogens, ethers, and esters.

Another embodiment comprises composition containing tryptanthrin andconjugates thereof.

Other embodiments relate to combinations of components. One embodimentrelates to compositions that include, as a first component, an activeingredient isolated or derived from an extract of hops and as a secondcomponent at least one member selected from the group consisting ofrosemary (Rosmarinus officinalis L.), an extract or compound derivedfrom rosemary, a triterpene species or derivatives or conjugatesthereof, and tryptanthrin or conjugates thereof. Another embodimentrelates to compositions that include, as a first component, tryptanthrinor conjugates thereof and as a second component at least one memberselected from the group consisting of an active ingredient isolated orderived from an extract of hops, rosemary, an extract or compoundderived from rosemary, and a triterpene species or derivatives orconjugates thereof.

Example 24 Effect of Modified Hops Component on NF-kB

As stated above, NF-κB, a heterodimer of the proteins p50 and RelA, isan inducible eukaryotic DNA binding protein complex that is broadlyexpressed and plays a pivotal role in regulating multiple biologicalresponses, such as the inflammatory and immune responses in mammaliancells. Targets of NF-κB include IL-2, the IL-2 receptor, and acute-phaseproteins of the liver. In addition to its role in immune responses,NF-κB activation overrides the apoptotic response to TNF and Fas,allowing for proliferation instead. NF-κB is cytoplasmic when inactive,maintained there by I-κB. As shown in FIG. 9, various stimuli lead toactivation of IKK (IκB Kinase), which phosphorylates IκB, marking it forubiquitination and degradation. Once IκB is degraded, NF-κB is freed toinitiate transcription. Following transcriptional activation of a gene,NF-κB is also rapidly degraded.

The ability to detect activated or nondegraded NF-κB is a function oftiming. Following cytokine stimulation of a cell, activated NF-κB can bedetected within 30 minutes. Within hours, the activated NF-κB isdegraded and only nonactivated, complexed NF-κB remains. In thisexample, whole cell NF-κB was determined 24 hours following trivalentstimuli of Interluken-1β (IL-1β), γ-interferon (IFN), and TNFα. By thistime, activated NF-κB has been degraded and only nonactivated, boundNF-κB remains. The IκB inhibitor is removed with lysis buffer and NF-κBis quantified by enzyme immunoassay following capture by dsDNAcontaining the NF-κB response element. Compounds or mixtures thatinhibit NF-κB activation can be identified as producing an increase inNF-κB-associated color development in cell lysates that have beentreated with cytokines and the test material. Since NF-κB plays a keyrole in regulating both inflammatory and immune responses in mammaliancells, as well as contributing to cancer cell growth and increasedreplication of various viruses like HIV-1, the development of agentsthat impair NF-κB activation or function could have importanttherapeutic applications.

Methods

Chemicals

NF-κB EIA kits were obtained from Active Motif (Carlsbad, Calif.). Heatinactivated Fetal Bovine Serum (FBS-HI Cat. #35-011CV), and Dulbeco'sModification of Eagle's Medium (DMEM Cat #10-013CV) was purchased fromMediatech (Herndon, Va.). Reduced isomerized-alpha acids (RIAA) wereobtained from John I. Haas, Inc., Yakima, Wash. Interluken-1β (IL-1β),γ-inteferon (IFN), TNFα, vitamin D3 (VD3) and all standard chemicalswere obtained from Sigma (St Louis, Mo.) and were of the highest puritycommercially available.

Cell Culture and Treatment of Cells

The human monocytic cell line U937 was obtained from the American TypeCulture Collection (Manasas, Va.) and subcultured according toinstructions from the supplier. The cells were routinely cultured at 37°C. with 5% CO₂ in RPMI 1640 (Life Technologies, Grand Island, N.Y.)containing 10% FBS, with 50 units penicillin/mL, 50 μg streptomycin/mL,5% sodium pyruvate, and 5% L-glutamine (Life Technologies). For theexperiment, U937 cells were cultured in 6-well plates at 37° C. with 5%CO₂ in a humidified incubator for 24 hours prior to treatment with testagents. RIAA in dimethylsulfoxide (10 μL) was added to the cells toachieve a final concentration of 10 μg RIAA/mL 60 min prior tostimulation with VD3 (100 nM) or VD3 and the cytokine mixture (25 ngIL-1β, 150 ng IFN, and 20 ng TNFa/mL). Twenty-four hr later, the cellswere washed and lysed with reagents supplied with the TransAM NFkB Chemikit.

Protein Assay

Protein concentrations of cell lysates were determined using theNanoOrange Protein Quantitation Kit with bovine serum albumin as thestandard (Molecular Probes, Eugene, Oreg.) according to the proceduresupplied by the manufacturer. Fluorescence was determined using aPackard FluoroCount, Model BF 10000 fluorometer with the excitationfilter set at 485 nm and emission filter set at 570 nm using PackardPlateReader version 3.0 software. The I-Smart program provided with thePackard PlateReader was used to calculate the protein concentration.

NF-κB Assay

The TransAM NFkB Chemi kit (Active Motiff) was used to detectnondegraded NF-κB p50 in the U937 cells. Instructions of the supplierwere followed with no modification. A Bio-tek Instruments ELISA platereader was used to record optical density at 405 nm. NF-κB wasquantified and tabulated as mOD₄₀₅ units.

Statistical Analysis

Means of four to eight replicates per treatment and 95% confidenceintervals were computed using standard statistical formula in Excelsspreadsheets (Microsoft, Redmond, Wash.).

Results

After 24 hr of stimulation with the cytokine cocktail, the amount ofnondegraded NF-κB in the U937 cells would be expected to decrease. Asshown in Table 16, controls (330 mOD units) and VD3 treatments containedapproximately twice the amount of NF-κB as those cells stimulated withthe cytokine cocktail or VD3 plus cytokine cocktail. The addition ofRIAA, however, prevented the activation and subsequent degradation ofNF-κB (281 vs 122 mOD units). This was a novel and unexpected finding.The lack of activation of NF-κB is favorable because NF-κB activationoverrides the apoptotic response to TNF and Fas and can cause a varietyof disorders. Also shown in Table 16, with RIAA alone, there was noactivation of the NF-κB complex. Therefore, hops components or modifiedhops components, such as RIAA, can serve to affect disorders associatedwith NF-κB activation since these components either do not activateNF-κB or prevent activation of NF-κB.

TABLE 16 NF-κB Treatment mOD units]† Control (Dimethylsulfoxide solventcontrols) 330 Vitamin D3 (100 nM) 391 (261-420)IL-1β/γ-interferon/TNFα(25/150/20 ng/mL) 167 (110-224) VD3 plusIL-1β/γ-interferon/TNFα 122 (79-165) Reduced isomerized alpha-acids(RIAA) 10 μg/mL) 362 (301-422) RIAA/VD3/IL-1β/γ-interferon/TNFα 281(241-321) †Parenthetic values are 95% confidence intervals.

In conclusion, it would be useful to identify a natural formulation ofcompounds that would to modulate NF-κB. Such a formulation haswidespread applications. It would also be useful to identify a naturalformulation of compounds that would inhibit expression of COX-2, inhibitprostaglandin synthesis selectively in target cells, or inhibitinflammation response selectively in target cells.

Example 25 Lack of Direct PGE₂ Inhibition by Reduced Isomerized AlphaAcids or Isomerized Alpha Acids in LPS-Stimulated RAW 264.7 Cells

This example describes testing of post COX-2 (cyclooxygenase-2)induction with A23187 arachidonic acid release.

The objective of this study was to assess the ability of the hopsderivatives RIAA (reduced isomerized alpha acids) (Redihop(rho-iso-alpha acids (RIAA), 29.5-30.5%, <0.2% iso-alpha acids) and IAA(isomerized alpha acids) (Isohop; iso-alpha acids (IAA), 29.5-30.5%) tofunction independently as direct inhibitors of COX-2 mediated PGE₂biosynthesis in the RAW 264.7 cell model of inflammation.

The following methods and procedures were used. Cell culture andtreatment with test material—RAW 264.7 cells (ATCC number TIB-71) wereobtained from the American Type Culture Collection (Manassas, Va.) andsub-cultured according to the instructions of the supplier. Inpreparation for testing, cells were grown in growth Dulbecco'sModification of Eagle's medium (DMEM) with 10% fetal bovine serum, heatinactivated (FBS-HI) with penicillin/streptomycin and maintained in logphase prior to experimental setup. On day two of the experiment, cellswere plated at 8×10⁴ cells per well in a 96-well tissue culture platewith 200 μL growth medium per well.

Following overnight incubation at 37° C. with 5% CO₂, the growth mediumwas aspirated and replaced with 200 μL DMEM with no FBS orpenicillin/streptomycin. RAW 264.7 cells were stimulated withlipopolysaccharide (LPS) (10 ng/ml final concentration) and incubatedovernight to induce COX-2 expression. Eighteen hours postLPS-stimulation, test material was added followed 60 minutes later bythe addition of A23187. Test materials were dissolved indimethylsulfoxide (DMSO) as a 250-fold stock solution. Four μL of this250-fold stock test material preparation was added to 1 mL of DMEM, and200 μL of this solution was added to eight wells for each dose of testmaterial. Supernatant media was sampled for prostaglandin E₂ (PGE₂)determination after 30 minutes. Median inhibitory concentrations werecomputed from a minimum of four concentrations over two independentexperiments. The combination indeces (CIs) were computed as describedbelow in statistical methods.

Table 17 describes the test materials used in each of two independentassays that were performed.

TABLE 17 Dosing matrix for LPS-stimulated RAW 264.7 cells followed bytreatment with test material TESTING FOR DIRECT COX-2 INHIBITINGACTIVITY PGE2 assay using undiluted and 1:20 dilution - 2 plates9.08.03-RAW 264.7 cells treated with LPS, incubated overnight thentreated with test material for 60 min followed by A23187 for 30 min C3C4 C5 C6 Compound Fraction RIAA [μg/mL] [μg/mL] [μg/mL] [μg/mL] No.Wells 1. Beta Tech RIAA 1.00 100 10 1.0 0.10 8 C7 C8 C9 C10 CompoundFraction IAA [μg/mL] [μg/mL] [μg/mL] [μg/mL] No. Wells 2. Beta Tech IAA1.00 100 10 1.0 0.10 8 C11 Aspirin 1.00 10 1.0 0.10 0.010 8 C12 APHS 101.0 0.10 0.010 8

Determination of PGE₂

A commercial, non-radioactive procedure for quantification of PGE₂ wasemployed (Caymen Chemical, Ann Arbor, Mich.) for the determination ofPGE₂ and the recommended procedure of the manufacturer was used withoutmodification. In summary, 50 μL of the supernatant culture medium werediluted with appropriate amounts of acetylcholinesterase-labeled tracerand PGE₂ antiserum, and incubated at room temperature for 18 h.Afterwards, the wells in the PGE₂-assay microtiter plate were emptiedand rinsed with wash buffer; two-hundred μL of Ellman's reagentcontaining substrate for acetylcholinesterase were then added. Thereaction was maintained on a slow shaker at room temperature for 1 h andthe absorbance at 415 nm was determined in a Bio-tek Instruments (Model#Elx800, Winooski, Vt.) enzyme-linked immunosorbant assay (ELISA) platereader. The manufacturer's specifications for this assay include anintra-assay coefficient of variation of <10%, cross reactivity with PGD₂and PGF₂ of less than 1% and linearity over the range of 10-1000 pgmL⁻¹. The PGE₂ concentration was computed as pg PGE₂ per 10⁵ cells.

Cell Viability

Cell viability was assessed by microscopic inspection of cells prior toor immediately following sampling of the medium for PGE₂ assay. Cellmortality was noted when observed.

The following materials were used and obtained by the indicatedmanufacturers. Bacterial lipopolysaccharide (LPS; B E. coli 055:B5) wasfrom Sigma (St. Louis, Mo.). Prostaglandin E₂ monoclonal antibody kitwas purchased from Cayman Chemical (Ann Arbor, Mich.). Heat inactivatedFetal Bovine Serum (FBS-HI Cat. #35-011CV) and Dulbecco's Modificationof Eagle's Medium (DMEM Cat #10-1013CV) was purchased from Mediatech(Herndon, Va.). Unless otherwise noted, all standard reagents wereobtained from Sigma (St. Louis, Mo.) and were the purest commerciallyavailable. Test substances included RIAA and IAA obtained from BetatechHops Products (Washington, D.C.).

The following statistical methods were used. A minimum of fourconcentrations (Table 1) was used to compute dose-response curves andmedium inhibitory concentrations (IC_(50s)) for PGE₂ with 95% confidenceintervals using CalcuSyn (BIOSOFT, Ferguson, Mo.). This statisticalpackage performs multiple drug dose-effect calculations using the MedianEffect methods described by T-C Chou and P. Talaly, Quantitativeanalysis of dose-effect relationships: the combined effects of multipledrugs or enzyme inhibitors. Adv Enzyme Regul 22, 27-55 (1984). Briefly,the analysis correlates the “Dose” and the “Effect” in the simplestpossible form: fa/fu=(C/C_(m))^(m), where C is the concentration or doseof the compound and Cm is the median-effective dose signifying thepotency. Cm is determined from the x-intercept of the median-effectplot. The fraction affected by the concentration of the test material isfa and the fraction unaffected by the concentration is fu (fu=1−fa). Theexponent m is the parameter signifying the sigmoidicity or shape of thedose-effect curve. It is estimated by the slope of the median-effectplot.

The median-effect plot is a graph of x=log(C) vs. y=log(fa/fu) and isbased on the logarithmic form of Chou's median-effect equation. Thegoodness of fit for the data to the median-effect equation isrepresented by the linear correlation coefficient r of the median-effectplot. Usually, the experimental data from enzyme or receptor systemshave an r>0.96, from tissue culture an r>0.90 and from animal systems anr>0.85. In the cell-based studies reported here, all linear correlationcoefficients were greater than 0.90. For most robust results,experiments are repeated a minimum of three times on three differentdates. The percent inhibition at each dose is averaged over the threeindependent experiments and used to calculate the median inhibitoryconcentrations reported.

Synergy of test components is quantified using the combination index(CI) parameter. The CI of Chou-Talaly is based on the multipledrug-effect and is derived from enzyme kinetic models (Chou, T.-C. andTalalay, P. A simple generalized equation for the analysis of multipleinhibitions of Michaelis-Menten kinetic systems. J. Biol. Chem.252:6438-6442 (1977). The equation determines only the additive effectrather than synergism or antagonism. However, synergism is defined inthis case as a more than expected additive effect, and antagonism as aless than expected additive effect as proposed by Cho and Talalay. Usingthe designation of CI=1 as the additive effect, the followingrelationships were obtained for mutually exclusive compounds that havethe same mode of action or for mutually non-exclusive drugs that havetotally independent modes of action: CI<1, =1, and >1 indicatingsynergism, additivity and antagonism, respectively.

Two data transformations were applied where warranted. The firsttransformation consisted of computing the percent inhibition from thehighest PGE₂ production produced from the lowest test concentration whenthe PGE₂ production of these low doses exceeded the PGE₂ production ofthe LPS-stimulated control. This process controls for responsevariability and gradients throughout the plate. The second datatransformation adjusted for variance in response at the graded doses.Monte Carlo simulations using the historical variance between wellspredicted that dose-response curves appear graded only 40% of the timewhen duplicate wells per concentration are used in a four-pointdose-response curve. Thus, sorting the response by concentration beforecalculating the IC₅₀ was done in those situations in which the responsedid not appear graded.

Using the protocol outlined above, LPS-stimulation of PGE₂ production inRAW 264.7 cells ranged from 1.4-fold to 2.1-fold relative tonon-stimulated cells and was somewhat dependent upon dilution of mediafor the PGE₂ assay. The IC₅₀ value of 8.7 μg/mL (95% CL (confidencelimit)=3.9-19) computed for the aspirin positive control was consistentwith published values for direct COX-2 inhibition ranging from 1.4 to 50μg/mL (Mitchell, J. A. et al. Selectivity of nonsteroidalanti-inflammatory drugs as inhibitors of constitutive and induciblecyclooxygenase. Proc. Natl. Acad. Sci. USA 90:11693-11697 (1994);Warner, T. D. et al. Nonsteroidal drug selectivities forcyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated withhuman gastrointestinal toxicity: A full in vitro analysis. Proc. Natl.Acad. Sci. USA 96:7563-7568 (1999)_(j) and previous results of 3.2 μg/mL(95% CL=0.55-19) in the A549 cell line.

RAW 264.7 cells were stimulated with LPS and incubated overnight toinduce COX-2 expression (FIG. 10). Eighteen hours post LPS-stimulation,test material was added followed 60 minutes later by the addition ofA23187. Supernatant media was sampled for PGE₂ determination after 30minutes. Mean percent PGE₂ inhibition values were computed from aminimum of eight replicates over four concentrations and two independentexperiments (FIG. 10).

Both RIAA and IAA produced modest, dose-related inhibition of PGE₂ inLPS-stimulated RAW 264.7 cells (FIG. 10). Over the 1000-fold increase inconcentration of test material, only a 14 and 10 percent increase ininhibition was noted, respectively, for RIAA and IAA. The shallowness ofthe dose-response slopes resulted in IC₅₀ values (Table 18) in the mg/mLrange for RIAA (36 mg/mL) and IAA (>1000 mg/mL). Such minimal change inresponse over three-log units of doses implies that the observedinhibitory effect of the hops derivatives in this cell-based assay islikely a secondary effect on the cells and not direct inhibition of COX.One possible explanation is that the hops derivatives interfere withA23187-mediated arachidonic acid release from cellular membranes.

RAW 264.7 cells were stimulated with LPS and incubated overnight toinduce COX-2 expression. Eighteen hours post LPS-stimulation, testmaterial was added followed 60 minutes later by the addition of A23187.Supernatant media was sampled for PGE₂ determination after 30 minutes.Median inhibitory concentrations were computed from a minimum of eightreplicates at four concentrations over two independent experiments(Table 18).

TABLE 18 Median inhibitory concentrations for RIAA, IAA in RAW 264.7cells when test material is added post overnight LPS-stimulation. IC₅₀95% Confidence Interval Test Material [mg/mL] [mg/mL] RIAA 36 17-79IAA >1000 — IC₅₀ 95% Confidence Interval Positive Control [μg/mL][μg/mL] Aspirin 8.7 μg/mL 3.9-19

The results of testing RIAA and IAA for their ability to directlyinhibit PGE₂ biosynthesis from COX-2 produced only a modest,dose-related inhibition of PGE₂ in LPS-stimulated RAW 264.7 cells. Theshallowness of the dose-response slopes resulted in IC₅₀ values in themg/mL range for RIAA (36 mg/mL) and IAA (>1000 mg/mL). The observedinhibitory effect of the hops derivatives in this cell-based assay islikely a secondary effect on the cells and not direct inhibition of COX.One possible explanation is that the hops derivatives interfere withA23187-mediated arachidonic acid release from cellular membranes.

Example 26 Analysis of Hops Activity in a Cell-Free COX Assay System

This example describes the effect of hops on COX enzyme activity.

The effect of hops on COX enzyme activity was tested in a cell-free COXassay system. The assay was performed using the Cayman Chemical COXInhibitor Screening Assay kit (Catalog #560131; Cayman Chemical Co.; AnnArbor Mich.). Briefly, enzyme was pre-incubated with inhibitor for tenminutes at 37° C., then the reaction was initiated with the addition ofarachidonic acid. After two minutes, the reaction was stopped by theaddition of HCl. The PGH₂ was reduced to PGH₂ alpha, which was thenquantified using a competitive enzyme immunoassay (EIA). Eachconcentration was tested with two separate reactions, which were theneach tested in duplicate during the EIA step.

The results of the COX enzyme assay are shown in Table 19. As can beseen, IAA and RIAA had essentially no effect on COX-1 or COX-2 enzymeactivity. In contrast, the COX inhibitor indomethacin inhibited bothCOX-1 and COX-2.

TABLE 19 IAA and RIAA inhibition of COX acitivity. COX 1 IndomethacinCOX 1 IAA COX 1 RIAA ug/ml % Inhibition ug/ml % Inhibition ug/ml %Inhibition 10 60.6 200 9.6 200 2.9 1 34.6 100 3 100 0.5 0.01 28.2 10 4.210 2.5 0.001 −2.8 1 2.1 1 1.9 COX 2 COX 2 COX 2 ug/ml % Inhibition ug/ml% Inhibition ug/ml % Inhibition 200 48.4 200 0.3 200 1.9 50 77.6 100−3.6 100 −0.7 0.5 67.5 10 −2 10 2.7 0.05 3.4 1 0.9 1 2.8

These results demonstrate that hops have no significant COX enzymeactivity. Therefore, hops likely acts at the level of expression ofCOX-2.

Example 27 Gastric Mucosal Cell Model for Estimating RelativeGastrointestinal Toxicity of NSAIDs

This example describes the use of a gastric mucosal cell line (AGScells) for determining potential gastrointestinal toxicity ofnon-steroidal anti-inflammatory drugs.

As described in Examples 1 and 2, AGS cells provide a model system fordetermining potential gastrointestinal toxicity. The objective of thisstudy was to further characterize the AGS human gastric mucosal cellline as a model for estimating relative GI toxicity (gastropathy) ofCOX-inhibiting compounds.

AGS cells were further characterized as a model for gastrointestinaltoxicity essentially as described in Example 2. Briefly, chemicals usedin the assays were obtained as follows. Commercial formulations ofrofecoxib tablets and celecoxib capsules were used. PGE₂ EIA kits wereobtained from Cayman Chemical (Ann Arbor, Mich.). Anti-COX-1 andanti-COX-2 rabbit polyclonal antisera were obtained from UpstateBiotechnology (Waltham, Mass.), and donkey anti-goat IgG-HRP wasprocured from Santa Cruz Biotechnology (Santa Cruz, Calif.). HeatInactivated Fetal Bovine Serum (FBS-HI Cat. #35-011CV) and Dulbecco'sModification of Eagle's Medium (DMEM Cat #10-013CV) was purchased fromMediatech (Herndon, Va.). Interleukin-1β (IL-1β) and all standardchemicals and non-steroidal anti-inflammatory drugs (NSAIDs), unlessnoted, were obtained from Sigma (St Louis, Mo.) and were of the highestpurity commercially available.

For cell culture, human airway epithelial cells A549 were obtained fromthe American Type Culture Collection (Manassas, Va.) and sub-culturedaccording to the instructions of the supplier. The cells were routinelycultured at 37° C. with 5% CO₂ in RPMI 1640 containing 10% FBS with 50units penicillin/mL, 50 μg streptomycin/mL, 5% sodium pyruvate, and 5%L-glutamine. For treatment of the cells with test compounds, the WilliamHarvey Modified Assay (WHMA) was used for determination of COX-2inhibition with no modifications [Warner T D, Giuliano F, Vojnovic I,Bukasa A, Mitchell J A, Vane J R. (1999) Non-steroidal drugselectivities for cyclooxygenase-1 rather than cyclooxygensase-2 areassociated with human gastrointestinal toxicity: A full in vitroanalysis. Proc. Natl. Acad. Sci. USA. 96:7563-7568]. Briefly, A549 cellswere exposed to IL-1β for 24 h, media was removed and human plasma (100μL) was added together with test agents or the DMSO vehicle. For initialexperiments, test agent concentrations were 25, 5, 0.5, and 0.05 μg/mL.DMSO in each microtiter well was less than 1% of the 200 μL volume; 60min later, A23187 (50 μM) was added; after 30 min, 50 μL of the culturesupernatant media was sampled and immediately assayed for PGE₂ asdetailed elsewhere. The AGS human gastric mucosal cell line (AmericanType Culture Collection, Manassas, Va.) was also cultured and maintainedaccording to recommended ATCC methodology. Sub-cultured AGS cells weregrown in IMDM with 20% FBS with 50 units penicillin/mL and 50 μgstreptomycin/mL; cells were maintained in log phase prior to eachexperiment. For PGE₂ assays, approximately 10⁵ cells per well wereplated into 96-well plates in 200 μL growth medium per well. Cells weregrown to 80% confluence and washed 3-times with IMDA media prior toaddition of test agent. NSAIDs were added in 200 μL of IMDA mediacontaining no FBS or penicillin/streptomycin. Sixty minutes followingaddition of the test materials, arachidonic acid (AA) was either inducedwith addition of the calcium ionophore A23187, or exogenously added at100 or 5 μM AA in DMSO. Incubation at 37° C. was carried out for anadditional 30 min. Fifty microliters of media were sampled for PGE₂determination.

For determination of PGE₂, a commercial, non-radioactive procedure forquantification of PGE₂ was employed (Cayman Chemical, Ann Arbor, Mich.)for the determination of PGE₂ and the recommended procedure of themanufacturer was used without modification. Briefly, 50 μL of thesupernatant culture medium, along with a serial dilution of PGE₂standard samples, were mixed with appropriate amounts ofacetylcholinesterase-labeled tracer and PGE₂ antiserum and incubated atroom temperature for 18 h. Afterwards, the wells in the PGE₂-assaymicrotiter plate were emptied and rinsed with wash buffer, and 200 μL ofEllman's reagent containing substrate for acetylcholinesterase were thenadded. The reaction was performed on a slow shaker at room temperaturefor 1 h, after which absorbance at 415 nm was determined in a Bio-TekInstrument ELISA plate reader (Model #Elx800, Winooski, Vt.). Themanufacturer's specifications for this assay include an intra-assaycoefficient of variation of <10%, cross reactivity with PGD₂ andPGF_(2α) of less than 1%, and linearity over the range of 10-1000 pg/mL.The PGE₂ concentration was recorded as pg PGE₂ per 10⁵ cells.

For assay calculations, a minimum of four concentrations each, with tworeplicates per concentration over three independent experiments, wereused to compute median inhibitory concentrations (IC₅₀) and their 95%confidence limits for the inhibition of PGE₂ biosynthesis (CalcuSyn,BIOSOFT, Ferguson, Mo.). Complete dose-response curves with coefficientsof determination >0.9 were obtained for all test compounds, exceptsalicylic acid and acetaminophen for A549 and AGS cells anddiisopropylfluorophosphate for AGS cells with 100 μM AA. The therapeuticindex (TI) for gastrointestinal safety was computed as the log (AGSIC₅₀/A549 IC₅₀). While it has been recommended that the IC₈₀ should beused for the efficacy component (i.e., inhibition of PGE₂ synthesis inA549 cells) in the calculation of the TI, large errors are associatedwith estimates at the extremes of the dose-response curve. Thus, thereexists greater uncertainty in ratios in which these estimates are used.Positive TI indicate low potential for GI toxicity, while negative TIindicate a higher potential for GI toxicity. Spearman's rank correlationcoefficient r_(s) was computed to quantify the degree of associationbetween ranking of previously published TI using non-target cells andthe AGS/A549 model. The parameter r_(s) was also used to determine thedegree of association between in vitro ranking of TI and ranking ofclinically assessed NSAID gastropathy. The probability of a Type I errorwas set at the nominal 5% level.

The AGS human gastric cell line was cultured in six-well plates at 37°C. with 5% CO2 in a humidified incubator for 24 h. Cells were lysed onice in lysis buffer and protein concentration determined. Fiftymicrograms of cell lysate were solubilized and fractionated on a 10%polyacrylamide gel containing sodium dodecylsulfate. For immunoblotting,Western blotting of COX-1 and COX-2 was performed using PAGEr™ GoldPrecast Gels (Bio Whittaker Molecular Applications, Rockland, Me.). RAW264.7 cell lysates containing approximately 60 μg protein were loadedwith laemmli sample buffer into the wells in a total volume of 30 μL.The vertical minigel electrophoresis chambers used were made by SavantInstruments, Inc. (Model MV 120; Holbrook, N.Y.). Gels were run at 40mA/plate (constant current) at room temperature until the bromophenolblue stain reached the bottom of the gel, which took about 1 h. Gelswere then blotted on polyvinyl fluoride transfer membranes (PVDF) (PallCorporation, Ann Arbor, Mich.), overnight, at 500 mA and 4° C. Molecularweight markers used were the unstained, broad-range Precision ProteinStandards (Bio Rad, Hercules, Calif.). The BioWest™ extended durationchemiluminescent substrate (BioImaging Systems, Upland, Calif.), anon-isotopic, horseradish peroxidase substrate kit for Western Blotdetection, was used for protein visualization. Images of Western Blotswere acquired using a UVP Epi Chemi II Darkroom (BioImaging Systems),and were analyzed and enhanced by LabWorks™ Image Acquisition andAnalysis Software (BioImaging Systems). Band intensities were evaluatedthrough densitometric analysis, computed using ScanAnalysis® software(BIOSOFT, Ferguson, Mo.), and recorded as arbitrary Density Units (DU).

The AGS cell line constitutively expressed both COX-1 and COX-2, withCOX-1 expression approximately 4 times greater than COX-2 expression(see Example 1). These results are in agreement with recently publishedwork (Fan et al., Interleukin-1beta induces cyclo-oxygenase-2 expressionin gastric cancer cells by the p38 and p44/42 mitogen-activated proteinkinase signaling pathways. J Gastroenterol Hepatol. 16:1098-104 (2001))using the AGS cell line, in which constitutive expression of bothcyclooxygenase enzymes was demonstrated.

Table 20 shows the median inhibitory concentration (IC50) for PGE2biosynthesis of select NSAID in the A549 and AGS cell model.

TABLE 20 Median inhibitory concentrations (IC₅₀) forPGE_(2 biosynthesis of) select NSAID in the A549 and AGS cell model.†AGS Gastric Mucosal Cells A549 100 μM 5 μM A23187 A23187 ArachidonateArachidonate Compounds [μM] [μM] [μM] [μM] DIFP††   6.5 359    >136   217  (1.5-28)   (125-1022) (141-185) Rofecoxib    0.24 5.5 12 21(0.15-0.45) (2.7-11)  (2.3-64)  (5.8-79)  Celecoxib    0.21  0.063 17  9.4 (0.01-4.2)  (0.02-0.22)  (2.5-113) (3.9-23)  Nimensulide    0.32 0.12 73 75 (0.16-0.65) (0.0081-1.7)    (25-211)  (52-104) Naproxen 28 0.83 167  313   (1.3-600) (0.24-2.8)   (16-1735)  (91-1039) Ibuprofen12 2.8   6.3 107  (6.8-19)  (1.3-5.8) (1.4-29)   (38-291) Aspirin 18 2.9  6.0 18  (3.0-106) (1.4-5.6) (2.9-12)  (9.4-37)  Salicylic acid 4246 1065    112  7848   (355-50971)   (94-12217)  (69-181)  (1775-34565)Acetaminophen 238  535    346  3815   (6.62-9589)  (179-1616) (192-609) (1152-12649) Indomethacin   8.1   0.0042     0.0025     0.0056(2.6-26)  (0.00042-0.039)  (0.000028-0.20)   (0.002-0.011) †Parentheticvalues are 95% confidence intervals of the IC₅₀ estimate. ††DIFP =diisofluorophosphate.

FIG. 11 shows a comparison of Log IC₅₀ ratios and ranking of potentialgastropathy. Log IC₅₀ ratios using the William Harvey Modified Assay(WHMA) are expressed WHMA COX-1/WHMA COX-2 from Warner, et al.(Nonsteroidal drug selectivities for cyclo-oxygenase-1 rather thancyclo-oxygensase-2 are associated with human gastrointestinal toxicity:A full in vitro analysis. Proc. Natl. Acad. Sci. USA.96:7563-7568(1999))(white bars) and Mitchell, et al. (Selectivity ofnonsteroidal antiinflammatory drugs as inhibitors of constitutive andinducible cyclooxygenase. Proc. Natl. Acad. Sci. USA. 90:11693-11697(1994))(blue bars) are shown in FIG. 11A, with log IC₅₀ ratios (AGS/WHMACOX-2) for AGS cells treated with A23187 FIG. 11B), 100 μM arachidonicacid (FIG. 11C), or 5 μM arachidonic acid (FIG. 11D). Values to theright of 0 indicate decreasing probability of gastropathy, whereasvalues to the left of 0 indicate increasing probability of gastropathy.

As shown in FIG. 11, the relationship between previously published dataand the three AGS protocols was examined. The log [IC₅₀ ratio (WHMACOX-1/WHMA COX-2)] was computed from published data (FIG. 11A) andcompared to the log [IC₅₀ ratios (AGS/WHMA COX-2)] for the A23187 (FIG.11B), 100 μM arachidonic acid (FIG. 11C), and 5 μM arachidonic acid(FIG. 11D) protocols. Ranking of compounds from lowest to greatest GItoxicity potential was strikingly similar among the models and withinAGS protocols. Interestingly, only rofecoxib and indomethacin showed theleast and most potential GI toxicity, respectively, in all AGSprotocols. Rank estimates of TI were similar to rank TI computed usingplatelets (COX-1) and A549 cells (COX-2) with r_(s)=0.903, p<0.01 forA23187; 0.733, p=0.02 for 100 μM AA; and 0.77, p<0.02 for 5 μM AA.Quantitatively, however, the AGS/A549 model exhibited lower TI than theplatelet/A549 model. A23187-mediated AA release, 100 μM AA addition, and5 μM AA addition provided rank estimates of GI toxicity significantlyassociated with clinical rankings of NSAID gastropathy, respectively(r_(s)=0.933, p<0.01; 0.783, p<0.01; 0.683, p=0.05). A23187 ranking ofNSAIDs from lowest to greatest potential for GI toxicity wasrofecoxib<acetaminophen<nimensulide<celecoxib<salicylicacid<ibuprofen<aspirin<naproxen<indomethacin.

As described in Example 1 and as seen in normal gastric mucosal cells,the AGS cell line constitutively expresses both COX-1 and COX-2. The useof AGS and A549 cells provided rank estimates of TI that werequalitatively similar to rank TI computed using platelets (COX-1) andA549 cells with r_(s)=0.903, p<0.01 for A23187; 0.733, p=0.02 for 100 μMAA; and 0.770, p<0.02 for 5 μM AA. Quantitatively, the AGS/A549 modelexhibited lower TI than the platelet/A549 model. A23187-mediated AArelease, as well as the 100 μM and 5 μM AA-addition protocols providedrank estimates of GI toxicity significantly associated with clinicalrankings of NSAID gastropathy, at r_(s)=0.933, p<0.01; 0.783, p<0.01;and 0.683, p=0.05, respectively. A23187 ranking of NSAIDs from lowest togreatest potential for GI toxicity wasrofecoxib<acetaminophen<nimensulide<celecoxib<salicylicacid<ibuprofen<aspirin<naproxen<indomethacin.

These results further demonstrate that the AGS human gastric mucosalcell line can serve as a model for assessing the gastrointestinaleffects of COX-inhibiting compounds. The AGS derived data indicate thatinhibition of gastric PGE₂ biosynthesis underlies the human gastropathyof NSAIDs.

A preferred embodiment comprises compositions containing at least onefraction isolated or derived from hops (Humulus lupulus). Examples offractions isolated or derived from hops are alpha acids, isoalpha acids,reduced isoalpha acids, tetra-hydroisoalpha acids, hexa-hydroisoalphaacids, beta acids, and spent hops. Preferred compounds of fractionsisolated or derived from hops, include, but are not limited to,humulone, cohumulone, adhumulone, isohumulone, isocohumulone,isoadhumulone, dihydro-isohumulone, dihydro-isocohumulone,dihydro-adhumulone, tetrahydro-isohumulone, tetrahydro-isocohumulone,tetrahydro-adhumulone, hexahydro-isohumulone, hexahydro-isocohumulone,and hexahydro-adhumulone. Preferred compounds can also bearsubstituents, such as halogens, ethers, and esters.

Another embodiment comprises composition containing tryptantin andconjugates thereof.

Other embodiments relate to combinations of components. One embodimentrelates to compositions that include, as a first component, an activeingredient isolated or derived from an extract of hops and as a secondcomponent at least one member selected from the group consisting ofrosemary (Rosmarinus officinalis L.), an extract or compound derivedfrom rosemary, a triterpene species or derivatives or conjugatesthereof, and tryptanthrin or conjugates thereof. Another embodimentrelates to compositions that include, as a first component, tryptanthrinor conjugates thereof and as a second component at least one memberselected from the group consisting of an active ingredient isolated orderived from an extract of hops, rosemary, an extract or compoundderived from rosemary, and a triterpene species or derivatives orconjugates thereof.

It will be readily apparent to those skilled in the art that variouschanges and modifications of an obvious nature may be made withoutdeparting from the spirit of the invention, and all such changes andmodifications are considered to fall within the scope of the inventionas defined by the appended claims. Such changes and modifications wouldinclude, but not be limited to, the incipient ingredients added toaffect the capsule, tablet, lotion, food or bar manufacturing process aswell as vitamins, herbs, flavorings and carriers. Other such changes ormodifications would include the use of other herbs or botanical productscontaining the combinations of the preferred embodiments disclosedabove. Many additional modifications and variations of the embodimentsdescribed herein may be made without departing from the scope, as isapparent to those skilled in the art. The specific embodiments describedherein are offered by way of example only.

What is claimed is:
 1. An anti-inflammatory composition comprisinganti-inflammatory effective amounts of a first component selected fromthe group consisting of dihydro-isohumulone, dihydro-isocohumulone,dihydro-isoadhumulone, tetrahydro-isohumulone, tetrahydro-isocohumulone,tetrahydro-isoadhumulone, hexahydro-isohumulone,hexahydro-isocohumulone, and hexahydro-isoadhumulone; and as a secondcomponent, at least one member selected from the group consisting ofrosemary, an extract derived from rosemary, a compound derived fromrosemary, and a triterpene species.
 2. The composition of claim 1,wherein the first component is derived from hops.
 3. The composition ofclaim 1, wherein the compound derived from rosemary is selected from thegroup consisting of 1,8-cineole, 19-alpha-hydroxyursolic acid,2-β-hydroxyoleanolic acid, 3-O-acetyloleanolic acid, 3-O-acetylursolicacid, 6-methoxy-luteolin-7-glucoside, 6-methoxyluteolin,6-methoxyluteolin-7-glucoside, methoxyluteolin-7-methylether,7-ethoxy-rosmanol, 7-methoxy-rosmanol, alpha-amyrin, alpha-humulene,alpha-hydroxyhydrocaffeic acid, alpha-pinene, alpha-terpinene,alpha-terpinenyl acetate, alpha-terpineol, alpha-thujone, apigenin,apigenin-7-glucoside, curcumene, benzyl-alcohol, β-amyrenone, β-amyrin,β-elemene, β-pinene, betulin, betulinic acid, borneol, bornyl-acetate,caffeic acid, camphene, camphor, carnosic acid, carnosol, carvacrol,carvone, caryophyllene, caryophyllene-oxide, chlorogenic acid,diosmetin, gamma-terpinene, hesperidin, isoborneol, limonene, luteolin,luteolin-3′-O-(3″-O-acetyl)-β-D-glucuronide,luteolin-3′-O-(4″-O-acetyl)-β-D-glucuronide,luteolin-3′-O-β-D-glucuronide, luteolin-7-glucoside, methyl-eugenol,myrcene, neo-chlorogenic acid, nepetin, octanoic acid, oleanolic acid,p-cymene, piperitenone, rosmanol, rosmaric acid, rosmaricine,rosmaridiphenol, rosemarinic acid, rosmarinol, rosmariquinone, sabinene,sabinyl acetate, salicylates, salicylic acid-2-β-D-glucoside, squalene,terpinen-4-ol, terpinolene, thymol, trans-anethole, trans-carveol,ursolic acid, verbenone, and zingiberene.
 4. The composition of claim 1,wherein the triterpene species is selected from the group consisting of18-a-glycyrrhetinic acid, 18-β-glycyrrhetinic acid,2-a-3-a-dihydrooxyurs-12-3n-28-onic acid, 3-a-hydroxyursolic acid,3-oxo-ursolic acid, betulin, betulinic acid, celastrol, eburicoic acid,friedelin, glycyrrhizin, gypsogenin, oleanolic acid, oleanolicacid-3-acetate, pachymic acid, pinicolic acid, sophoradiol,soyasapogenol A, soyasapogenol B, tripteris, triptophenolide, tumulosicacid, ursolic acid, ursolic acid-3-acetate, uvaol, and β-sitosterol. 5.The composition of claim 1, wherein the composition comprises about 0.5to 10,000 mg of the first component.
 6. The composition of claim 1,wherein the composition comprises about 0.5 to 5,000 mg of the secondcomponent, and wherein the second component is selected from the groupconsisting of rosemary, an extract derived from rosemary, and a compoundderived from rosemary.
 7. The composition of claim 1, wherein thecomposition comprises about 0.035 to 3,500 mg of the triterpene species.8. The composition of claim 1, wherein the composition comprises about0.001 to 10 weight percent of the first component.
 9. The composition ofclaim 1, wherein the composition comprises about 0.001 to 10 weightpercent of the second component.
 10. The composition of claim 1, whereina ratio of the first component to the second component is in the rangeof about 100:1 to about 1:100.
 11. The composition of claim 1, whereinthe composition further comprises a pharmaceutically acceptable carrier.12. The composition of claim 1, further comprising glucosamine.